Biglycan and related therapeutics and methods of use

ABSTRACT

The invention provides compositions and methods for treating, preventing, and diagnosing diseases or conditions associated with an abnormal level or activity of biglycan; disorders associated with an unstable cytoplasmic membrane, due, e.g., to an unstable dystrophin associated protein complex (DAPC); disorders associated with abnormal synapses or neuromuscular junctions, including those resulting from an abnormal MuSK activation or acetylcholine receptor (AChR) aggregation. Example of diseases include muscular dystrophies, such as Duchenne&#39;s Muscular Dystrophy, Becker&#39;s Muscular Dystrophy, neuromuscular disorders and neurological disorders.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 12/498,172 filedJul. 6, 2009 now U.S. Pat. No. 8,138,154, which is a divisional of U.S.Ser. No. 10/081,736 filed Feb. 20, 2002 now U.S. Pat. No. 7,612,038,which is a continuation-in-part of U.S. Ser. No. 09/715,836 filed Nov.17, 2000 and now issued as U.S. Pat. No. 6,864,236, which claims thebenefit of U.S. Provisional Application No. 60/166,253, filed Nov. 18,1999. U.S. Ser. No. 10/081,736 filed Feb. 20, 2002 also claims thebenefit of U.S. Provisional Application No. 60/270,053, filed Feb. 20,2001. The specifications of each application are specificallyincorporated herein.

GOVERNMENT GRANTS

This invention was made with government support under Grants HD23924 andMH53571 awarded by the National Institutes of Health. The government hascertain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted via EFS-Web and is hereby incorporated by reference in itsentirety. Said ASCII copy, created on May 29, 2012, is namedBURFP07006seq.txt and is 8,906 bytes in size.

BACKGROUND OF THE INVENTION

The dystrophin-associated protein complex (DAPC) links the cytoskeletonto the extracellular matrix and is necessary for maintaining theintegrity of the muscle cell\ plasma membrane. The core DAPC consists ofthe cytoskeletal scaffolding molecule dystrophin and the dystroglycanand sarcoglycan transmembrane subcomplexes. The DAPC also serves tolocalize key signaling molecules to the cell surface, at least in partthrough its associated syntrophins (Brenman, et al. (1996) Cell. 84:757-767; Bredt, et al. (1998), Proc Natl Acad Sci USA. 95: 14592).Mutations in either dystrophin or any of the sarcoglycans result inmuscular dystrophies characterized by breakdown of the muscle cellmembrane, loss of myofibers, and fibrosis (Hoffman, et al. 1987. Cell.51: 919; Straub, and Campbell (1997) Curr Opin Neurol. 10: 168).Moreover, mutations in the extracellular matrix protein laminin-α2,which associates with the DAPC on the cell surface, is the basis of amajor congenital muscular dystrophy (Helbling-Leclerc, et al. (1995)Nat. Genet. 11: 216).

The α-/β-dystroglycan subcomplex forms a critical structural link in theDAPC. The transmembrane β-dystroglycan and the wholly extracellularα-dystroglycan arise by proteolytic cleavage of a common precursor(Ibraghimov, et al. (1992) Nature 355: 696; Bowe, et al. (1994) Neuron12: 1173). The cytoplasmic tail of β-dystroglycan binds dystrophin,while the highly glycosylated, mucin-like α-dystroglycan binds toseveral ECM elements including agrin, laminin, and perlecan (Ervasti andCampbell, (1993) J Cell Biol. 122: 809; Bowe, et al. (1994) Neuron. 12:1173; Gee, et al. (1994) Cell 77: 675; Hemler, (1999) Cell 97: 543).This binding to matrix proteins appears to be essential for assembly ofbasal lamina, since mice deficient in dystroglycan fail to form thesestructures and die very early in development (Henry, M. D. and K. P.Campbell. 1998. Cell. 95: 859). β-Dystroglycan can bind the signalingadapter molecule Grb2 and associates indirectly with p125FAK (Yang, etal. (1995) J. Biol. Chem. 270: 11711; Cavaldesi, et al. (1999), J.Neurochem. 72: 01648). Although the significance of these associationsremains unknown, these binding properties suggest that dystroglycan mayalso serve to localize signaling molecules to the cell surface.

Several lines of evidence suggest that dystroglycan may also function inneuromuscular junction formation, in particular, in postsynapticdifferentiation. For purposes of clarity, the components of theneuromuscular junction are summarized here. The major structuralfeatures of the neuromuscular junction (NMJ) or nerve-muscle synapse arethe pre- and post-synaptic specializations of the motor neuron andmuscle, respectively, the intervening synaptic basal lamina, and thespecialized Schwann cell cap (Salpeter, et al (1987) The VertebrateNeuromuscular Junction. New York, Alan R. Liss.). The presynapticapparatus is marked by ordered arrays of synaptic vesicles, a subset ofwhich are poised to fuse with the plasma membrane at the active zones,and release acethylcholine that is recognized by acetylcholine receptors(AChRs) on the muscle, and ultimately results in electrical activationand contraction of the muscle (Heuser, et al (1981) J. Cell Biol. 88:564) Immediately across the 50 nm synaptic cleft from these zones arethe crests of the postjunctional folds. These crests bristle withAcetylcholine receptors (AChRs), which can reach densities of >10,000molecules/μm² (Fertuck, et al (1976) J. Cell. Biol. 69: 144). Thelocalized and tightly regulated secretion of acetylcholine into thenarrow synaptic cleft, coupled with the high AChR density in thepostsynaptic membrane, ensures rapid and reliable synaptic transmissionbetween neuron and muscle. Perturbations of these specializations, suchas the decrease in the number of functional AChRs seen in myastheniagravis, can lead to debilitating and often fatal clinical outcomes(Oosterhuis, et al (1992) Neurology & Neurosurgery 5: 638).

The synaptic basal lamina (SBL) is interposed between the pre- andpost-synaptic membranes and contains molecules important for thestructure, function, and regulation of the neuromuscular junction (Bowe,M. A & Fallon, J. R., (1995) Ann. Rev. Neurosci. 18: 443; Sanes, et al.(1999) Ann. Rev. Neurosci. 22: 389). It consists of a distinct set ofextracellular matrix molecules including specialized laminins,proteoglycans and collagens (Hall, et al (1993) Neuron 10: (Suppl.) 99).The SBL also contains molecules essential for the regulation of synapticstructure and function including AChE, neuregulins, and agrin. The SBLthus serves both as a specialized structure for maintaining thelocalized differentiation of the synapse as well as a repository foressential regulatory molecules.

The molecular composition of the postsynaptic membrane is known inconsiderable detail. As noted above, the most abundant membrane proteinis the AChR. The cytosolic AChR associated protein rapsyn (formerlyknown as the 43kD) protein) is present at stoichiometric levels with thereceptor and is likely to form a key link between the cytosolic domainof the AchR and the cytoskeleton (Froehner, et al (1995) Nature 377:195; Gautam, et al. (1995) Nature 377: 232). The postsynaptic membraneis also enriched in erbB2-4, some or all of which serve as neuregulinreceptors (Altiok, et al. (1995) EMBO J. 14: 4258; Zhu, et al. (1995)EMBO J. 14: 5842). AChR and other molecules essential for nerve-musclecommunication. The cytoskeletal elements can be broadly grouped into twosubsets. Dystrophin and utrophin are members of thedystrophin-associated protein complex, or DAPC, and are linked to thesynaptic basal lamina via the transmembrane heteromer α-/β-dystroglycan.The postsynaptic cytoskeleton is also enriched in several focaladhesion-associated molecules including α-actinin, vinculin, talin,paxillin, and filamin (Sanes, et al (1999) Ann. Rev. Neurosci. 22: 389).The latter proteins probably communicate, directly or indirectly, withthe extracellular matrix through integrins, some of which are enrichedat synapses (Martin, et al. (1996) Dev. Biol. 174: 125). Actin isassociated with both sets of cytoskeletal molecules (Rybakova et al.(1996) J. Cell Biol. 135: 661; Amann, et al. (1998) J. Biol. Chem. 273:28419-23; Schoenwaelder et al. (1999) Curr. Opin. Cell. Biol. 11: 274).The functions of these specialized sets of proteins are consideredbelow.

α-Dystroglycan binds the synapse organizing molecule agrin (Bowe, et al.(1994) Neuron. 12: 1173; Campanelli, et al. (1994) Cell. 77: 663; Gee,et al. (1994) Cell. 77: 675; Sugiyama, et al. (1994) Neuron. 13: 103;O'Toole, et al. (1996) Proc Natl Acad Sci USA. 93: 7369) (reviewed inFallon and Hall, (1994) Trends Neurosci. 17: 469), and β-dystroglycanbinds to the AChR-associated protein rapsyn (Cartaud, et al. (1998) JBiol Chem. 273: 11321). Further, agrin-induced AChR clustering on thepostsynaptic membrane is markedly decreased in muscle cells expressingreduced levels of dystroglycan (Montanaro, et al. (1998) J Neurosci. 18:1250). The precise role of dystroglycan in this process is unknown.Currently available evidence suggests that dystroglycan is not part ofthe primary agrin receptor, but rather may play a structural role in theorganization of postsynaptic specializations (Gesemann, et al. (1995)Biol. 128: 625; Glass, et al. (1996) Cell. 85: 513; Jacobson, et al.(1998) J Neurosci. 18: 6340).

Another molecule that plays an important role in neuromuscular junctionformation is the tyrosine kinase receptor MuSK, which becomesphosphorylated in response to agrin. However, agrin does not bind toMuSK and it is unclear how agrin stimulates MuSK. The existence of aco-receptor had been suggested. Activation of MuSK by antibodycross-linking is sufficient to induce the clustering of AChRs oncultured myotubes (Xie et al. (1997) Nat. Biotechnol. 15:768 and Hopfand Hoch (1998) J. Biol. Chem. 273: 6467) and a constitutively activeMuSK can induce postsynaptic differentiation in vivo (Jones et al.(1999) J. Neurosci. 19:3376). However, MuSK phosphorylation is necessarybut not sufficient for agrin-induced AChR clustering.

The realm of dystroglycan function ranges far beyond muscle. As notedabove, mice defective in dystroglycan die long before muscledifferentiation. In a surprising development, α-dystroglycan innon-muscle cells has been shown to function as a receptor for LassaFever and choriomeningitis fever viruses (Cao, W., et al., 1998,Science. 282: 2079), and on Schwann cells as a co-receptor forMycobacterium leprae (Rambukkana, et al. (1998) Science. 282: 2076).Dystroglycan is also abundant in brain, but its function there is notunderstood (Gorecki, et al. (1994) Hum Mol Genet. 3: 1589; Smalheiserand Kim (1995) J Biol. Chem. 270: 15425).

α-Dystroglycan is comprised of three known domains. An amino-terminaldomain folds into an autonomous globular configuration (Brancaccio, etal. (1995) Febs Lett. 368: 139). The middle third of the protein isserine- and threonine-rich, and is highly glycosylated (Brancaccio, etal. (1997) Eur J Biochem. 246: 166). Indeed, the core molecular weightof α-dystroglycan is ˜68 kDa, but the native molecule migrates onSDS-PAGE as a polydisperse band whose size ranges from 120-190 kDa,depending upon the species and tissue source (Ervasti and Campbell(1993) J Cell Biol. 122: 809; Bowe, et al. (1994) Neuron. 12: 1173; Gee,et al. (1994) Cell. 77: 675; Matsumura, et al. (1997) J Biol. Chem. 272:13904). Glycosylation of α-dystroglycan, probably in this middle third,is essential for its laminin- and agrin-binding properties.

While it is clear that dystroglycan and the DAPC play crucial roles in avariety of processes in muscle as well as in other tissues, theunderlying mechanisms remain obscure.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for stabilizingdystrophin-associated protein complexes (DAPCs) on the surface of acell. Stabilizing DAPC complexes on cell membranes allows membranes tobe less “leaky” and thus, provides a longer life span to cells. Theinvention also provides methods for activating a postynaptic membrane,such as to render the membrane more sensitive to an incoming signal froma neural cell (e.g., at a neuromuscular junction). Activating apostsynaptic membrane may comprise stimulating aggregation of AChR onthe cell membrane and/or activating MuSK, such as by phosphorylation.

In one embodiment, the method comprises contacting the target cell witha biglycan polypeptide comprising an amino acid sequence which is atleast about 90% identical to the biglycan sequence of SEQ ID NO: 9 or aportion thereof. In a preferred method, the biglycan polypeptide bindsto α-dystroglycan; α-sarcoglycan and/or γ-sarcoglycan. In an even morepreferred embodiment, the biglycan polypeptide stimulatesphosphorylation of α-sarcoglycan on a cell membrane. The biglycanpolypeptide also preferably potentiates agrin-induced AChR aggregationon the surface of the cell; stimulate the phosphorylation of MuSK on thecell; and/or potentiates agrin-induced phosphorylation of MuSK.

The biglycan polypeptide may comprise one or more 24 amino acids repeatmotifs in the Leucine Rich Repeat (LRR) of human biglycan having SEQ IDNO: 9. In another embodiment, the biglycan polypeptide comprises acysteine-rich region, e.g., the C-terminal or the N-terminalCysteine-rich region. The biglycan polypeptide may include one or moreglycosaminoglycan (GAG) chains. In an even more preferred embodiment,the biglycan polypeptide comprises an amino acid sequence which is atleast about 90% identical to amino acids 20-368 or 38-368 of SEQ ID NO:9, even more preferably at least 95% identical or 100% identical toamino acids 20-368 or 38-368 of SEQ ID NO: 9. In another embodiment, thebiglycan polypeptide is encoded by a nucleic acid which hybridizes toSEQ ID NO: 8. The biglycan polypeptide can be Torpedo DAG-125, or thehuman biglycan of SEQ ID NO: 9, or a portion thereof having at least onebiological activity of biglycan.

In other embodiments, the biglycan therapeutic is a peptide fragment ofthe full length protein. Preferably it is a fragment which retains theability to induce phosphorylation of sarcoglycans and upregulateutrophin activity/expression. For instance, a preferred peptide fragmentbinds to and activates MuSK.

In other embodiments, the subject biglycan therapeutics arepeptidomimetics of a portion of a biglycan protein. Peptidomimetics arecompounds based on, or derived from, peptides and proteins. The biglycanpeptidomimetics of the present invention typically can be obtained bystructural modification of a known biglycan peptide sequence usingunnatural amino acids, conformational restraints, isosteric replacement,and the like. The subject peptidomimetics constitute the continuum ofstructural space between peptides and non-peptide synthetic structures;biglycan peptidomimetics may be useful, therefore, in delineatingpharmacophores and in helping to translate peptides into nonpeptidecompounds with the activity of the parent biglycan peptides.

Moreover, as is apparent from the present disclosure, mimetopes of thesubject biglycan peptides can be provided. Such peptidomimetics can havesuch attributes as being non-hydrolyzable (e.g., increased stabilityagainst proteases or other physiological conditions which degrade thecorresponding peptide), increased specificity and/or potency, andincreased cell permeability for intracellular localization of thepeptidomimetic. For illustrative purposes, peptide analogs of thepresent invention can be generated using, for example, benzodiazepines(e.g., see Freidinger et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988), substitutedgama lactam rings (Garvey et al. in Peptides: Chemistry and Biology, G.R. Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p123), C-7mimics (Huffman et al. in Peptides: Chemistry and Biology, G. R.Marshall ed., ESCOM Publisher: Leiden, Netherlands, 1988, p. 105),keto-methylene pseudopeptides (Ewenson et al. (1986) J Med Chem 29:295;and Ewenson et al. in Peptides: Structure and Function (Proceedings ofthe 9th American Peptide Symposium) Pierce Chemical Co. Rockland, Ill.,1985), β-turn dipeptide cores (Nagai et al. (1985) Tetrahedron Lett26:647; and Sato et al. (1986) J Chem Soc Perkin Trans 1:1231),β-aminoalcohols (Gordon et al. (1985) Biochem Biophys Res Commun126:419;and Dann et al. (1986) Biochem Biophys Res Commun 134:71),diaminoketones (Natarajan et al. (1984) Biochem Biophys Res Commun124:141), and methyleneamino-modified (Roark et al. in Peptides:Chemistry and Biology, G. R. Marshall ed., ESCOM Publisher: Leiden,Netherlands, 1988, p134). Also, see generally, Session III: Analytic andsynthetic methods, in Peptides: Chemistry and Biology, G. R. Marshalled., ESCOM Publisher: Leiden, Netherlands, 1988)

In addition to a variety of sidechain replacements which can be carriedout to generate the subject biglycan peptidomimetics, the presentinvention specifically contemplates the use of conformationallyrestrained mimics of peptide secondary structure. Numerous surrogateshave been developed for the amide bond of peptides. Frequently exploitedsurrogates for the amide bond include the following groups (i)trans-olefins, (ii) fluoroalkene, (iii) methyleneamino, (iv)phosphonamides, and (v) sulfonamides.

Examples of Surrogates

Additionally, peptidomimietics based on more substantial modificationsof the backbone of the biglycan peptide can be used. Peptidomimeticswhich fall in this category include (i) retro-inverso analogs, and (ii)N-alkyl glycine analogs (so-called peptoids).

Examples of Analogs

Furthermore, the methods of combinatorial chemistry are being brought tobear, c.f. Verdine et al. PCT publication WO9948897, on the developmentof new peptidomimetics. For example, one embodiment of a so-called“peptide morphing” strategy focuses on the random generation of alibrary of peptide analogs that comprise a wide range of peptide bondsubstitutes.

The invention also provides a method for treating or preventing acondition associated with an abnormal dystrophin-associated proteincomplex (DAPC) in cells of a subject, comprising administering to thesubject a pharmaceutically efficient amount of a biglycan polypeptide,peptide or peptidomimetic or a biglycan agonist (collectively referredto herein as “biglycan therapeutics”) which stabilizes the DAPC.Examples of diseases that can be treated or prevented include musculardystrophies, such as Duchenne's Muscular Dystrophy, Becker's MuscularDystrophy, Congenital Muscular Dystrophy, Limb-girdle MuscularDystrophy, and mytonic dystrophy; and cardiomyopathies.

In another example, the invention provides a method for treating orpreventing a condition characterized by an abnormal neuromuscularjunction or synapse in a subject, comprising administering to thesubject a pharmaceutically efficient amount of a biglycan therapeuticwhich binds to, and/or induces phosphorylation of MuSK and/or whichinduces aggregation of acetylcholine receptors (AChRs). The conditioncan be a neuromuscular or neurological disease.

The invention also provides methods for treating, preventing anddiagnosing diseases or disorders that are associated with abnormallevels or activity of biglycan; with unstable cytoplasmic membranes, duein particular, to unstable DAPCs; or abnormal synapses or neuromuscularjunctions.

In yet another example, the invention provides a diagnostic method fordetermining whether a subject has or is at risk of developing acondition associated with an abnormal DAPC or abnormal synapse orneuromuscular junction, or other disease associated with an abnormalbiglycan level or activity, comprising determining the level or activityof biglycan in a tissue of the subject, wherein the presence of anabnormal level and/or activity of biglycan in the tissue of a subjectindicates that the subject has or is at risk of developing a conditionassociated with an abnormal DAPC or abnormal synapse or neuromuscularjunction or other disease associated with an abnormal biglycan level oractivity.

Also within the scope of the invention are screening methods foridentifying agents with inhibit or potentiate the activity of biglycan,such as a human biglycan or Torpedo DAG-125, such as agents whichpotentiate or inhibit biglycan binding to another molecule, such as amember of a DAPC or MuSK. Agents identified in these assays can be used,e.g., in therapeutic methods, as biglycan therapeutics. Screeningmethods for identifying agents which modulate phosphorylation induced bybiglycan are also within the scope of the invention.

Other aspects of the invention are described below or will be apparentto those skilled in the art in light of the present disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a diagram of the interaction between DAG-125 or biglycan witha DAPC.

FIG. 2 shows the results of a ligand blot overlay assay, in whichfilters with various extracts (as indicated) were incubated withportions of α-dystroglycan.

FIG. 3 (A-C) shows the results of a blot overlay assays in which filterswith input and elutes from columns were incubated with portions of alphadystroglycan or agrin.

FIG. 4 is a diagram showing portions of dystroglycan used in a blotoverlay assays and the presence (+) or absence (−) of binding.

FIG. 5A shows a blot overlay assay in which a filter with synapticmembranes, input or elute from a column was incubated with a portion ofalpha-dystroglycan.

FIG. 5B shows the sequence alignment between the Torpedo DAG-125sequences (SEQ ID NOs: 1-3) and human biglycan (SEQ ID NOs: 4-6). FIG.5C is a diagram of the structure of biglycan: the prepro-region, whichis absent in the mature biglycan corresponds to amino acids 1-37 of SEQID NO: 9; the N-terminal cysteine-rich region corresponds to amino acids38-80 of SEQ ID NO: 9; the LLR region corresponds to about amino acids81-314 of SEQ ID NO: 9; and the C-terminal cysteine-rich regioncorresponds to amino acids 315-368 of SEQ ID NO: 9. Circles representchondroitin sulfate side chains. “S—S” denotes intrachain disulfidebinding.

FIG. 6 shows the results of an analysis of Torpedo DAG-125glycosylation.

FIG. 7 shows that the binding of dystroglycan to biglycan is dependentupon specific chondroitin sulfate side chains. QE-Bgn is bacteriallyexpressed biglycan core. AC stands for articular cartilage.

FIG. 8A-C show overlay assays blots containing biglycan proteoglycan(BGN-PG), biglycan core (BGN), a biglycan-decorin hybrid (Hybrid),decorin proteoglycan (DEC-PG), decorin (DEC), bacterially producedbiglycan (QE-BIG), and Torpedo electric organ membrane fraction (TEOM),which were incubated with ³⁵S labeled α-sarcoglycan (FIG. 8A),γ-sarcoglycan (FIG. 8B), and delta-sarcoglycan (FIG. 8C).

FIG. 9 shows biglycan expression at the neuromuscular junction.

FIG. 10 shows the upregulation of biglycan expression in wild type (wt)and dystrophic (mdx) muscle.

FIG. 11 shows the results of a co-immunoprecipitation of biglycan withrecombinant MuSK-Fc.

FIG. 12 is a Western blot containing cell extracts of cells incubatedwith or without agrin and with biglycan proteoglycan (BGNPG) or decoringproteoglycan (DECPG) incubated with anti-phosphotyrosine antibody.

FIG. 13A shows a genotype analysis. PCR genotyping was performed ongenomic DNA using primer pairs specific for mutant and wild typebiglycan alleles (Xu et al. 1998). PCR products from a wild type (male;+/o), a heterozygote (female; +/−), and a knockout (male; −/o) areshown. Size of PCR products is indicated on left.

FIG. 13B shows defective agrin-induced AChR clustering in myotubescultured from biglycan null mice and its rescue by addition of exogneousbiglycan. A Bgn female (+/−) was mated to a Bgn male (+/o) and primarycultures were established from each male pup in the resulting litter.The genotype of each pup was determined as shown in FIG. 13A. Myotubecultures derived from each mouse were then treated either with orwithout recombinant agrin 4.8 for 18 hours. Myotubes were then labeledwith rhodamine-a-bungarotoxin to visualize AChRs. Wild type myotubesshow a robust AChR clustering response to agrin, while myotubes frombiglycan−/o mice fail to cluster AChR in reponse to agrin. Exogenousbiglycan (1.4 nM) restores the agrin-induced AChR clustering response.

FIG. 13C shows quantification of AChR clustering. AChR clusters andmyotubes were counted in a minimum of 10 fields for cultures treatedeither with (AGRIN) or without (Con) recombinant agrin4.8 in thepresence of biglycan (1.4 nM) as indicated. A similar deficit inagrin-induced AChR clustering was observed in two other experiments.

FIG. 14 shows the level of serum creatine kinase in wild type andbiglycan knock out mice.

FIG. 15. Exogenous biglycan induces α-sarcoglycan phosphorylation in aMuSK dependent manner. Wild type C2C12 myotubes (lanes 1, 2, and 6) andMuSK null myotubes (lanes 3-5) were treated for thirty minutes asfollows: lanes 1, 3, and 6, unstimulated; lanes 2 and 5, stimulated witha mixture of recombinant proteoglycan and core biglycan (produced inosteosarcoma cells; 1 mg/mL); lane 4, stimulated with agrin 12.4.8. Thecultures were detergent extracted and α-sarcoglycan wasimmunoprecipitated, separated by SDS-PAGE, blotted, and probed withanti-phosphotyrosine antibody (lanes 1-5) or MIgG (lane 6). The additionof biglycan induced tyrosine phosphorylation of α-sarcoglycan and p35 inwild type C2C12 cells but not in MuSK knockout cells.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The invention is based at least in part on the observation that biglycaninteracts with, and regulates and/or induces modification of thedystrophin-associated protein complex (DAPC), as well as activatescomponents playing an important role in neuromuscular junctionformation. In particular, biglycan is shown to interact withα-dystroglycan, an extracellular component of the DAPC, as well as withα-sarcoglycan and γ-sarcoglycan, which are components of the sarcogycancomplex of the DAPC. Biglycan is also shown to induce phosphorylationα-sarcoglycan, showing that biglycan does not solely interact withcomponents of the DAPC, but also causes modification of the components.The proteoglycan of the invention has been found to be overexpressed inan animal model of muscular dystrophy that is characterized by theabsence of dystrophin. The integrity of the DAPC and its associationwith the extracellular matrix (ECM) are essential for muscle cellviability. Accordingly, biglycan is believed to stabilize the DAPCcomplex at the surface of cells, in particular, muscle cells, and can bepart of a compensatory mechanism that allows survival of dystrophinnegative fibers.

It has also been shown herein that biglycan is involved in neuromuscularjunction formation, e.g., induced by agrin. Agrin, which is anextracellular matrix protein present in the synaptic basal lamina, issecreted by the nerve terminal and triggers neuromuscular junctionformation by activating the receptor tyrosine kinase MuSK, therebyinducing phosphorylation and clustering of AChR. It had not previouslybeen known how agrin activates the receptor MuSK, since agrin does notbind directly to this receptor. As described below, activation of thereceptor MuSK by agrin is actually potentiated by biglycan. Thisdiscovery is based at least in part on the finding that biglycan bindsdirectly to the MuSK receptor; biglycan directly induces the tyrosinephosphorylation of MuSK; biglycan potentiates agrin-inducedphosphorylation of MuSK; and biglycan potentiates agrin-inducedclustering of AChRs. In addition, the appended examples demonstrate thatmyotubes from biglycan deficient mice show a defective response toagrin, in particular the cells are defective in agrin-induced AChRclustering, which was further shown to be corrected by the addition ofbiglycan to the culture media of the myotubes. Thus, it is clearly shownthat the absence of biglycan in cells results in a deficiency inagrin-induced AChR clustering, which can be corrected by the ectopicaddition of biglycan to the cells. The role of biglycan in mediatingneuromuscular junction formation, in particular, postynapticdifferentiation, is further supported by the fact biglycan binds toα-dystroglycan (shown herein), and that α- and β-dystroglycans interactwith components of the postsynaptic membrane. For example, agrin bindsto α-dystroglycan (see FIG. 1) and β-dystroglycan binds to theAChR-associated protein rapsyn. In addition, agrin-induced AChRclustering is markedly decreased in muscle cells expressing reducedlevels of dystroglycan, further demonstrating the role of dystroglycanin postsynaptic membranes. Thus, it was demonstrated herein thatbiglycan plays an important role in the formation of neuromuscularjunctions both by interacting with the agrin receptor MuSK and byinteracting with α-dystroglycan. It is contemplated that biglycan playsboth functional and structural roles in the organization of thepostsynaptic specializations.

Moreover, as described further below, biglycan also regulaties utrophinexpression and localization. Agrin can cause an upregulation of utrophinexpression and direct it to be localized to specific domains on the cellsurface. The signaling receptor for agrin is the receptor tyrosinekinase MuSK. Agrin also induces the tyrosine phosphorylation of α- andγ-sarcoglycan in cultured myotubes. Biglycan can also regulate thetyrosine phosphorylation of α- and γ-sarcoglycan. Moreover, the receptortyrosine kinase MuSK is required for this biglycan-induced tyrosinephosphorylation of these proteins. These observations indicate thatbiglycan can act directly to organize the DAPC, including utrophin, onthe muscle cell surface.

Furthermore, since DAPCs are also found in brain, agrin has been foundin senile plaques in brains of subjects with Alzheimer's disease, andperipheral and central neural deficiencies are present in some patientslacking dystrophin, biglycan is also believed to be involved information of synapses.

Thus, the results described herein indicate that biglycan plays animportant role in maintaining the integrity of muscle cell plasmamembrane, at least in part by interacting with α-dystroglycan and thesarcoglycans in the DAPC; in neuromuscular junction formation, at leastin part by mediating agrin-induced AChR clustering and MuSK activation;and also probably in synapse formation. Based at least on thesefindings, the invention provides compositions and methods fordiagnosing, treating and/or preventing diseases or conditions associatedwith a dysfunctional DAPC, an unstable cellular structure, a defect inneuromuscular junctions or synapses. Such diseases include, inparticular, muscular dystrophies, such as Duchenne, Limb-girdle, othermyopathies, neuromuscular disorders, and neurological disorders.

Furthermore, in view of the wide tissue distribution of DAPCs anddystroglycans, biglycan is likely to play a role in regulating signalingthrough the cytoplasmic membrane and/or maintaining the integrity ofcytoplasmic membranes of cells other than muscle cells. For example,dystroglycan or other DAPC components are abundant in brain, kidney, andheart. Thus, the invention provides, more generally, compositions,diagnostic and therapeutic methods for diseases or disorders associatedwith an abnormality of a membrane protein complex with which the proteinof the invention interacts, e.g., the DAPC, or MuSK receptor.

Based at least on the fact that dystroglycan is known to be a receptorused by microorganisms for entering cells, e.g., Lassa Fever andchoriomeningitis fever viruses, the compositions of the invention,particularly biglycan therapeutics, can be used for treating and/orpreventing infections by such microorganisms. Without wanting to belimited to a specific mechanism of action, biglycan therapeutics mayhinder or inhibit binding of the microorganism to dystroglycan.

Both human biglycan (described, e.g., in Fischer et al. as “bone smallproteoglycan” J. Biol. Chem. 264: 4571 (1996); GenBank Accession No.J04599; SEQ ID NO: 9) and DAG-125 isolated from Torpedo electric organhave been shown to interact with DAPC components. Based on sequencehomologies between the two proteins and similar biological activities(further described herein), it is believed that the human biglycan (SEQID NO: 9) may be the human ortholog of the Torpedo DAG-125.Alternatively, the human ortholog of the Torpedo DAG-125 may be aprotein that is highly related to human biglycan. For purposes ofclarity, the term “biglycan” as used herein is intended to include thehuman biglycan (SEQ ID NO: 9) and Torpedo DAG-125, as well as homologsof these proteoglycans.

II. Definitions

For convenience, the meaning of certain terms and phrases employed inthe specification, examples, and appended claims are provided below.

“GAGs” refers to glycosaminoglycans, which is used interchangeablyherein with “mucopolysaccharides,” are long, unbranched polysaccharidechains composed of repeating disaccharide units. One of the two sugarsis always an amino sugar (N-acetylglucosamine or N-acetylgalactosamine)Glycosaminoglycans are covalently linked to a serine residue of a coreprotein, to form a proteoglycan molecule.

The term “glycan” is used interchangeably herein with the term“polysaccharide” and “oligosaccharide.”

The term “glycoprotein” refers to a protein which contains one or morecarbohydrate groups covalently attached to the polypeptide chain.Typically, a glycoprotein contains from 1% to 60% carbohydrate by weightin the form of numerous, relatively short, branched oligosaccharidechains of variable composition. In contrast to glycoproteins,proteoglycans are much larger (up to millions of daltons), and theycontain 90% to 95% carbohydrate by weight in the form of may long,unbranched glycosaminoglycan chains.

The term “proteoglycan of the invention” refers to a proteoglycanmolecule having one or more of the characteristics and biologicalactivities of biglycan. Accordingly, a preferred proteoglycan of theinvention includes a proteoglycan having one or more of the followingcharacteristics: a molecular weight between 100 and 150 kDa, or anapparent mobility of 125 kDa, as determined on an SDS acrylamide gel;one or more glycosaminoglycan side chain; a molecular weight of the corebetween 35 and 40 kDa, preferably around 37 kDa; an amino acid sequenceselected from SEQ ID NO: 1-6 and 9 or variant thereof; one of morebiological activities of biglycan, as listed infra, under thecorresponding definition. In one embodiment, the proteoglycan of theinvention is a SLRP, e.g., human biglycan. A preferred proteoglycan ofthe invention is Torpedo DAG-125 or a mammalian, preferably human,ortholog thereof. Another preferred proteoglycan of the invention isbiglycan, e.g., human biglycan having SEQ ID NO: 9. The term“proteoglycan of the invention” further includes portions of thewildtype proteoglycan, provided that these portions have at least onebiological activity of a biglycan protein. Accordingly, the term“proteoglycan of the invention” includes molecules that consist only ofthe core (i.e., protein part of the molecule), or of the GAG sidechains, portions thereof and/or combinations thereof.

The term “biglycan” refers to proteoglycans having at least onebiological activity of human biglycan or Torpedo DAG-125. Preferredbiglycans include Torpedo DAG-125 (comprising SEQ ID NO: 1-3), humanbiglycan (SEQ ID NO: 9), as well as homologs and fragments thereof.Preferred homologs are proteoglycans or proteins or peptides having atleast about 70% identity, at least about 75% identity, at least about80% identity, at least about 85% identity, at least about 90% identity,at least about 95% identity, and even more preferably, at least about 98or 99% identity. Even more preferred homologs are those which have acertain percentage of homology (or identity) with human biglycan orTorpedo DAG-125 and have at least one biological activity of theseproteoglycans. The term biglycan is not limited to the full lengthbiglycan, but includes also portions having at least one activity ofbiglycan.

The term “human biglycan” refers to the proteoglycan described inFischer et al. J. Biol. Chem. 264: 4571 (1989), having GenBank AccessionNo. J04599, and the amino acid sequence set forth in SEQ ID NO: 9. AcDNA sequence encoding the human biglycan protein is set forth in SEQ IDNO: 7, and the open reading frame thereof as SEQ ID NO: 8.

The term “biglycan core” refers to a biglycan that does not include GAGchains.

The term “biglycan proteoglycan” or “biglycan PG” refers to a biglycanhaving at least one GAG chain.

The term “biglycan nucleic acid” refers to a nucleic acid encoding abiglycan proteoglycan, e.g., a nucleic acid encoding a protein havingSEQ ID NO: 9.

A “biological activity of biglycan” is intended to refer to one or moreof: the ability to maintain the integrity of a plasma membrane; theability to stabilize DAPCs on plasma membranes; the ability to bind toone or more components of DAPCs; e.g., binding to α-dystroglycan,binding to a sarcoglycan component, such as α-sarcoglycan orγ-sarcoglycan; binding to MuSK; stimulating the formation ofneuromuscluar junctions, such as by stimulating postsynapticdifferentiation; potentiation of AChR aggregation, e.g., agrin-inducedAChR aggregation; phosphorylation of DAPC components, e.g.,sarcoglycans; stimulation MuSK phosphorylation or potentiatingagrin-induced MuSK phosphorylation.

A “biglycan therapeutic” is a compound which can be used for treating orpreventing a disease that is associated with an abnormal cytoplasmicmembrane, e.g., an unstable membrane; an abnormal DAPC; abnormalneuromuscular junction; abnormal synapse; abnormal AChR aggregation; orabnormal MuSK activation. A biglycan therapeutic can be an agonist or anantagonist of one or more of the biological activities of biglycan. Atherapeutic can be any type of compound, including a protein orderivative thereof, e.g., a proteoglycan, a nucleic acid, a glycan, or asmall organic or synthetic molecule.

The term “abnormal” is used interchangeably herein with “aberrant” andrefers to a molecule, or activity with differs from the wild type ornormal molecule or activity.

The term “DAPC” refers to “dystrophin-associated protein complex”, amembrane complex, set forth in FIG. 1, which comprises dystrophin, α-and betα-dystroglycans, and the sarcoglycan transmembrane complex.

“Sarcoglycans” exit in different forms including α-, beta-, γ-, delta-,and epsilon-sarcoglycans. Certain sarcoglycans are specific for certaintissues, e.g., alpha and delta-sarcoglycans are skeletal musclespecific.

“Dystrophin-associated proteins” includes proteins or glycoproteins,such as alpha-dystroglycan, dystrobrevin, sarcospan and the syntrophins.

The term “AChR” refers to acetylcholine receptor.

The term “SLRP” refers to small leucine rich repeat proteoglycan.

The term “MASC” refers to muscle cell-associated specificity component.

The term “RATL” refers to rapsyn-associated transmembrane linker.

The term “HSPG” refers to heparan sulfate proteoglycans.

The term “MuSK” used interchangeably herein with “muscle specifickinase,” refers to a protein tyrosine kinase, that is expressed innormal and denervated muscle, as well as other tissues including heart,spleen, ovary or retina (See Valenzuela, D., et al., 1995, Neuron 15:573-584). The tyrosine kinase has alternatively been referred to as“Dmk” for “denervated muscle kinase.” Thus, the terms MuSK and Dmk maybe used interchangeably. The protein appears to be related to the Trkfamily of tyrosine kinases, and is further described in U.S. Pat. No.5,814,478.

The term “MuSK activating molecule” as used herein refers to a moleculewhich is capable of inducing phosphorylation of the MuSK receptor in thecontext of a differentiated muscle cell. One such activating molecule isagrin as described in the Examples set forth herein.

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides: “reference sequence”, “comparisonwindow”, “sequence identity”, “percentage of sequence identity”, and“substantial identity”. A “reference sequence” is a defined sequenceused as a basis for a sequence comparison; a reference sequence may be asubset of a larger sequence, for example, as a segment of a full-lengthcDNA or gene sequence given in a sequence listing, such as apolynucleotide sequence of SEQ ID NO: 7 or 8, or may comprise a completecDNA or gene sequence. Generally, a reference sequence is at least 20nucleotides in length, frequently at least 25 nucleotides in length, andoften at least 50 nucleotides in length. Since two polynucleotides mayeach (1) comprise a sequence (i.e., a portion of the completepolynucleotide sequence) that is similar between the twopolynucleotides, and (2) may further comprise a sequence that isdivergent between the two polynucleotides, sequence comparisons betweentwo (or more) polynucleotides are typically performed by comparingsequences of the two polynucleotides over a “comparison window” toidentify and compare local regions of sequence similarity. A “comparisonwindow”, as used herein, refers to a conceptual segment of at least 20contiguous nucleotide positions wherein a polynucleotide sequence may becompared to a reference sequence of at least 20 contiguous nucleotidesand wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) of 20 percent orless as compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two sequences.Optimal alignment of sequences for aligning a comparison window may beconducted by the local homology algorithm of Smith and Waterman (1981)Adv. Appl. Math. 2: 482, by the homology alignment algorithm ofNeedleman and Wunsch (1970) J. Mol. Biol. 48: 443, by the search forsimilarity method of Pearson and Lipman (1988) Proc. Natl. Acad. Sci.(U.S.A.) 85: 2444, by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage Release 7.0, Genetics Computer Group, 575 Science Dr., Madison,Wis.), or by inspection, and the best alignment (i.e., resulting in thehighest percentage of homology over the comparison window) generated bythe various methods is selected. The term “sequence identity” means thattwo polynucleotide sequences are identical (i.e., on anucleotide-by-nucleotide basis) over the window of comparison. The term“percentage of sequence identity” is calculated by comparing twooptimally aligned sequences over the window of comparison, determiningthe number of positions at which the identical nucleic acid base (e.g.,A, T, C, G, U, or I) occurs in both sequences to yield the number ofmatched positions, dividing the number of matched positions by the totalnumber of positions in the window of comparison (i.e., the window size),and multiplying the result by 100 to yield the percentage of sequenceidentity. The terms “substantial identity” as used herein denotes acharacteristic of a polynucleotide sequence, wherein the polynucleotidecomprises a sequence that has at least 85 percent sequence identity,preferably at least 90 to 95 percent sequence identity, more usually atleast 99 percent sequence identity as compared to a reference sequenceover a comparison window of at least 20 nucleotide positions, frequentlyover a window of at least 25-50 nucleotides, wherein the percentage ofsequence identity is calculated by comparing the reference sequence tothe polynucleotide sequence which may include deletions or additionswhich total 20 percent or less of the reference sequence over the windowof comparison. The reference sequence may be a subset of a largersequence, for example, as a segment of the full-length human biglycanpolynucleotide sequence.

As applied to polypeptides, the term “substantial identity” means thattwo peptide sequences, when optimally aligned, such as by the programsGAP or BESTFIT using default gap weights, share at least 80 percentsequence identity, preferably at least 90 percent sequence identity,more preferably at least 95 percent sequence identity or more (e.g., 99percent sequence identity). Preferably, residue positions which are notidentical differ by conservative amino acid substitutions. Conservativeamino acid substitutions refer to the interchangeability of residueshaving similar side chains. For example, a group of amino acids havingaliphatic side chains is glycine, alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Preferred conservative amino acidssubstitution groups are: valine-leucine-isoleucine,phenylalanine-tyrosine, lysine-arginine, alanine-valine, andasparagine-glutamine.

“Small molecule” as used herein, is meant to refer to a composition,which has a molecular weight of less than about 5 kD and most preferablyless than about 4 kD. Small molecules can be nucleic acids, peptides,polypeptides, peptidomimetics, carbohydrates, lipids or other organic(carbon containing) or inorganic molecules. Many pharmaceuticalcompanies have extensive libraries of chemical and/or biologicalmixtures, often fungal, bacterial, or algal extracts, which can bescreened with any of the assays of the invention to identify compoundsthat modulate the bioactivity of a proteoglycan of the invention.

A “myoblast” is a cell, that by fusion with other myoblasts, gives riseto myotubes that eventually develop into skeletal muscle fibres. Theterm is sometimes used for all the cells recognisable as immediateprecursors of skeletal muscle fibres. Alternatively, the term isreserved for those post-mitotic cells capable of fusion, others beingreferred to as presumptive myoblasts.

“Myofibril” is a long cylindrical organelle of striated muscle, composedof regular arrays of thick and thin filaments, and constituting thecontractile apparatus.

A “myotube” is an elongated multinucleate cells (three or more nuclei)that contain some peripherally located myofibrils. They are formed invivo or in vitro by the fusion of myoblasts and eventually develop intomature muscle fibres that have peripherally located nuclei and most oftheir cytoplasm filled with myofibrils. In fact, there is no very cleardistinction between myotubes and muscle fibers proper.

“Utrophin” (dystrophin associated protein) is an autosomal homologue ofdystrophin (of size 395 kD) localised near the neuromuscular junction inadult muscle, though in the absence of dystrophin (i.e. in Duchennemuscular dystrophy) utrophin is also located on the cytoplasmic face ofthe sarcolemma.

As used herein, the term “transfection” means the introduction of anucleic acid, e.g., an expression vector, into a recipient cell bynucleic acid-mediated gene transfer. The term “transduction” isgenerally used herein when the transfection with a nucleic acid is byviral delivery of the nucleic acid. “Transformation”, as used herein,refers to a process in which a cell's genotype is changed as a result ofthe cellular uptake of exogenous DNA or RNA, and, for example, thetransformed cell expresses a recombinant form of a polypeptide or, inthe case of anti-sense expression from the transferred gene, theexpression of a naturally-occurring form of the recombinant protein isdisrupted.

As used herein, the term “transgene” refers to a nucleic acid sequencewhich has been introduced into a cell. Daughter cells deriving from acell in which a transgene has been introduced are also said to containthe transgene (unless it has been deleted). A transgene can encode,e.g., a polypeptide, partly or entirely heterologous, i.e., foreign, tothe transgenic animal or cell into which it is introduced, or, ishomologous to an endogenous gene of the transgenic animal or cell intowhich it is introduced, but which is designed to be inserted, or isinserted, into the animal's genome in such a way as to alter the genomeof the cell into which it is inserted (e.g., it is inserted at alocation which differs from that of the natural gene). Alternatively, atransgene can also be present in an episome. A transgene can include oneor more transcriptional regulatory sequences and any other nucleic acid,(e.g. intron), that may be necessary for optimal expression of aselected coding sequence.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting another nucleic acid to which it has beenlinked. One type of preferred vector is an episome, i.e., a nucleic acidcapable of extra-chromosomal replication. Preferred vectors are thosecapable of autonomous replication and/or expression of nucleic acids towhich they are linked. Vectors capable of directing the expression ofgenes to which they are operatively linked are referred to herein as“expression vectors”. In general, expression vectors of utility inrecombinant DNA techniques are often in the form of “plasmids” whichrefer generally to circular double stranded DNA loops which, in theirvector form are not bound to the chromosome. In the presentspecification, “plasmid” and “vector” are used interchangeably as theplasmid is the most commonly used form of vector. However, the inventionis intended to include such other forms of expression vectors whichserve equivalent functions and which become known in the artsubsequently hereto.

“Derived from” as that phrase is used herein indicates a peptide ornucleotide sequence selected from within a given sequence. A peptide ornucleotide sequence derived from a named sequence may contain a smallnumber of modifications relative to the parent sequence, in most casesrepresenting deletion, replacement or insertion of less than about 15%,preferably less than about 10%, and in many cases less than about 5%, ofamino acid residues or base pairs present in the parent sequence. In thecase of DNAs, one DNA molecule is also considered to be derived fromanother if the two are capable of selectively hybridizing to oneanother.

The terms “chimeric”, “fusion” and “composite” are used to denote aprotein, peptide domain or nucleotide sequence or molecule containing atleast two component portions which are mutually heterologous in thesense that they are not, otherwise, found directly (covalently) linkedin nature. More specifically, the component portions are not found inthe same continuous polypeptide or gene in nature, at least not in thesame order or orientation or with the same spacing present in thechimeric protein or composite domain. Such materials contain componentsderived from at least two different proteins or genes or from at leasttwo non-adjacent portions of the same protein or gene. Compositeproteins, and DNA sequences which encode them, are recombinant in thesense that they contain at least two constituent portions which are nototherwise found directly linked (covalently) together in nature.

The term “modulate” refers to inhibiting or stimulating.

The terms “activating a postsynaptic membrane” refers to the stimulationof the transfer of a signal at neuromuscular junction, generally, from anerve cell to a muscle cell. Activation usually includes the stimulationof aggregation of AChR on the cell membrane at the neuromuscularjunction; and/or the phosphorylation of MuSK. Activation results ininduction of postsynaptic differentiation.

The term “treating” with regard to a subject, refers to improving atleast one symptom of the subject's disease or disorder. Treating can becuring the disease or condition or improving it, but reducing at leastcertain symptoms of it.

III. Compounds of the Invention

One aspect of the invention provides biglycan therapeutics for use inmaintaining the integrity of plasma cell membranes, in particular,biglycan therapeutics which stabilize dystrophin associated proteincomplexes (DAPC) in these membranes, thereby preventing thedisintegration of the membranes. The invention also provides biglycantherapeutics which stimulate neuromuscular junction formation, such asby stimulating postsynaptic membrane differentiation, and more generallycompounds which stimulate synapse formation.

In a particular embodiment, the biglycan therapeutics binds to one ormore components of the DAPC. The compound preferably binds toα-dystroglycan and/or to a sarcoglycan component, such as α-sarcoglycan.In an even more preferred embodiment, the compound of the inventionbinds both to α-dystroglycan and to a component of the sarcoglycancomplex, e.g., selected from the group consisting of α-sarcoglycan,γ-sarcoglycan and δ-sarcoglycan. The component of the sarcoglycan towhich the compound of the invention binds is preferably α-sarcoglycan.Generally, the compound of the invention contacts one or more componentsof the DAPC, e.g., to thereby stabilize the complex and reducedestabilization of the plasma membrane resulting from an abnormal DAPCcomplex, such as those seen in muscular dystrophies.

Yet in an even more preferred embodiment, the compound of the inventionbinds to a region of α-dystroglycan which is different from the regionto which agrin, laminin and perlecan bind (see FIG. 1). Binding of thecompounds of the invention do not require the presence of glycosyl sidechains on α-dystroglycan. More preferably, the compounds of theinvention bind to the C-terminal part of α-dystrogylcan, preferably toabout amino acids 345 to 891, more preferably to about amino acids1-750, about amino acids 30-654, about amino acids 345-653, or aboutamino acids 494-653 of human alpha-dystroglycan. Thus, a preferredcompound of the invention binds to a region consisting essentially ofthe C-terminal 150 amino acids of α-dystroglycan, i.e., amino acids494-653.

Other biglycan therapeutics of the invention bind to the receptortyrosine kinase MuSK. Such compounds can bind to MuSK and/orα-dystroglycan and/or a component of the sarcoglycan complex, e.g.,α-sarcoglycan. In preferred embodiments, the biglycan therapeuticactivates MuSK and induces phosphorylation of α and/or γ-sarcoglycan.

The subject biglycan therapeutics preferably bind specifically to one ormore of the above-cited molecules, i.e., they do not significantly or ata detectable level bind to other molecules to produce an undesirableeffect in the cell. The compounds preferably bind with a dissociationconstant of 10⁻⁶ or less, and even more preferably with a dissociationconstant of 10⁻⁷, 10⁻⁸, 10⁻⁹, 10⁻¹⁰, 10⁻¹¹, 10⁻¹², 10⁻¹², or 10⁻¹³ M orless. The dissociation constant can be determined according to methodswell known in the art.

Binding assays for determining the level of binding of a compound to acomponent of the DAPC or to MuSK or for identifying members of, e.g., alibrary of compounds which bind to these molecules are known in the artand are also further described herein. Methods for preparing DAPCcomponents or MuSK for use in such assays are also known. Suchcomponents can be isolated from tissue or, when they are proteins, canbe prepared recombinantly or synthetically. Their nucleotide and aminoacid sequences are publicly available, e.g., from GenBank, or frompublications.

Other preferred compounds of the invention have one or more biologicalactivities of biglycan, in addition to, or instead of, being able tobind one or more components of the DAPC and/or MuSK. For example, acompound of the invention can stimulate neuromuscular junctionformation, in particular, postsynatic membrane differentiation,including inducing aggregation of AChRs and/or stimulating orstimulating agrin-induced tyrosine phorphorylation of MusK.

The compound of the invention can be a protein or derivative thereof, inparticular a proteoglycan, a nucleic acid, such as a nucleic acidencoding a proteoglycan of the invention, a glycan, a peptidomimetic orderivative thereof, or a small organic molecule. Generally, the compoundcan be any type of molecule provided that the compound has the requiredcharacteristics, e.g., binding to α-sarcoglycan and/or other DAPCcomponents.

In a preferred embodiment, the compound of the invention is aproteoglycan having a molecular weight from about 100 kDa to about 150kDa, preferably from about 110 kDa to about 140 kDa, and most preferablyfrom about 120 to about 130 kDa, as determined, e.g., by migration on anSDS acrylamide gel. The core of the proteoglycan of the invention has amolecular weight from about 25 to about 45 kDa, preferably from about 30to about 40 kDa and most preferably around 37 kDa. Fragments or portionsof these proteoglycans are also within the scope of the invention.

The proteoglycan preferably contains one or more glycosaminoglycan sidechains, such as a mucopolysaccharide side chain, e.g., heparan,chondroitin, or dermatan. Preferred side chains consist of chonddroitinsulfate, e.g., 4-sulfate (chondroitin sulfate type A) and 6-sulfate(chondroitin sulfate type C). Any side chain can be used in theinvention, provided that the proteoglycan has at least one bioactivityof biglycan.

In an even more preferred embodiment, the proteoglycan of the inventioncomprises one or more of the following amino acid sequence in its core:IQAIEFEDL (SEQ ID NO: 1); LGLGFNEIR (SEQ ID NO: 2); andTSYHGISLFNNPVNYWDVL (SEQ ID NO: 3), or amino acid sequences relatedthereto, such as amino acid sequences from the mammalian ortholog of theTorpedo protein from which these amino acid sequences were obtained. Theproteoglycan preferably contains all three of these sequences orsequences related thereto. For example, the proteoglycan of theinvention can comprise one or more of the following amino acidsequences, which are part of human biglycan: IQAIELEDL (SEQ ID NO: 4);LGLGHNQIR (SEQ ID NO: 5); and AYYNGISLFNNPVPYWEVQ (SEQ ID NO: 6).

Although composition including, and methods using, Torpedo DAG-125 arewithin the scope of the invention, preferred compositions and methodsare those relating to mammalian, including vertebrate, homologs ofTorpedo DAG-125, referred to herein as orthologs of Torpedo DAG-125.Preferred orthologs of Torpedo DAG-125 are human, rodent, murine,canine, feline, ovine, and bovine orthologs. As shown herein, it ishighly likely that the mammalian DAG-125 is biglycan, however, it mayalso be a molecule that is related to biglycan, and, e.g., also todecorin (see below), but is actually a not previously described protein.Thus, the invention also provides compositions comprising the mammalianortholog of Torpedo DAG-125, such as the human ortholog of TorpedoDAG-125.

A mammalian ortholog of Torpedo DAG-125 can be isolated by screeninglibraries with probes containing nucleotide sequences encoding one ormore of SEQ ID NOs 1-3. Numerous other methods are available for cloningthe mammalian ortholog of Torpedo DAG-125. For example, antibodies toTorpedo DAG-125 can be produced and used to screen mammalian expressionlibraries. The identification of the cloned proteins as mammalianortholgogs of Torpedo DAG-125 can be established by performing the samebiological assays as thos described in the Examples employing TorpedoDAG-125.

Thus, the proteoglycan of the invention can also be a member of thefamily of small leucine-rich proteoglycans (SLRP), also referred to as“nonaggreagating or small dermatan-sulfate proteoglycans because oftheir inability to interact with hyaluronan, or because of their type ofglycosaminoglycans, respectively. SLRPs are organized into three classesbased on their protein and genomic organization. All SLRPs arecharacterized by a central domain containing leucine rich repeats (LRR)flanked at either side by small cysteine clusters. The SLRPs aredescribed, e.g., in Iozzo et al. (1998) Ann. Rev. Biochem. 67:609,specifically incorporated herein by reference.

SLRP protein cores range from ˜35-45 kD with one or two GAG chainsattached at the extreme N-terminus. The general structure of the SLRPprotein core consists of a tandem array of 6-10 leucine-rich repeats(LRR) flanked by domains with conserved, disulfide-bonded cysteines(FIG. 5C). Depending upon the extent of glycosylation and number of GAGchains, the native molecular weight ranges from ˜100-250 kD. On thebasis of their sequence homology, Iozzo, supra, has proposed that SLRPsbe grouped into three classes consisting of: 1) biglycan and decorin; 2)fibromodulin, lumican, keratocan, PREPLP, and osteoadherin; and 3)epiphycan and osteoglycin. The most compelling feature of the SLRPprotein core are the LRRs. Such repeats (24aa each in the SLRPs) mediateprotein-protein interactions in a wide variety of intracellular,transmembrane, and extracellular contexts (Kobe & Deisenhofer, (1994)Trends Biochem. Sci. 19: 415-21). The neurotrophin binding site on trkB,for example, is an LRR (Windisch et al., (1995) Biochemistry 34:11256-63). The repeats are thought to have a general structure of anα-helix followed by beta-sheet in an anti-parallel array, althoughsequence analysis has suggested that this order might be reversed in theSLRPs (Hocking et al., (1998) Matrix Biol. 17: 1-19). It is likely thatthe conserved residues of each repeat dictate their secondary structure,while the intervening amino acids determine specificity of ligandbinding.

Preferred SLRPs for use in the invention include Class I SLRPs, such asbiglycan and decorin. The partial amino acid sequences of DAG-125, theTorpedo proteoglycan which was shown to bind to alpha-dystroglycan (seeExamples) shows strong homology to human biglycan (see FIG. 5B): a 78%identity was found in a total of 37 amino acid long sequence. Biglycanfrom rodent, pig and human are >95% identical. Decorin and biglycan fromhuman are only 55% identical. Such homology is consistent with decorinand biglycan having both shared and unique functions. Thus, althoughTorpedo DAG-125 has amino acid sequence that more closely resemble thatof human biglycan, based on the similarity of structure and functionbetween biglycan and decorin, the latter proteoglycan and derivativesthereof may also be used to practice the invention.

Nucleotide and amino acid sequences of biglycan and decorin genes andproteins from various species are publically available, such as inGenBank. For example, human biglycan can be found under GenBankAccession No. J04599 (human hPGI encoding bone small proteoglycan I(biglycan), described in Fisher et al. (1989) J. Biol. Chem. 264: 4571;SEQ ID Nos: 7-9) and M65154; cow biglycan can be found under GenBankAccession No. L07953; rat biglycan can be found under GenBank AccessionNo. U17834, mouse biglycan can be found under GenBank Accession No.L20276 and X53928; ovis biglycan can be found under GenBank AccessionNo. AF034842; human decorin can be found at GenBank Accession No.M14219; rabbit decorin can be found at GenBank Accession No. I47020;chick decorin can be found at GenBank Accession No. P28675; Equusdecorin can be found at GenBank Accession No. AF038; bovine decorin canbe found at GenBank Accession No. P21793; ovis decorin can be found atGenBank Accession No. AF125041; and rat decorin can be found at GenBankAccession No. Q01129. Sequences of biglycan and decorin and other SLRPscan be found in GenBank.

Decorin and biglycan have one and two glycosaminoglycan (GAG) chains,respectively. Their composition is tissue specific and can be regulatedat a number of levels (Hocking et al., (1998) Matrix Biol 17: 1-19). Forexample, the biglycan GAG from skin and cartilage is predominantlydermatan sulfate, while biglycan synthesized in bone is a chondroitinsulfate proteoglycan. Heparan sulfate side chains have not beenreported. Both the protein core and the cell type contribute to thedistinct glycosylation of these SLRPs.

Other proteoglycans or cores thereof of the invention include fusionproteins. For example, biglycan or a portion thereof can be fused to animmunoglobulin portion. Alternatively, the fusion protein is acombination between two or more portions of proteoglycans of theinvention, e.g., a portion of a biglycan molecule fused to a portion ofa decorin molecule (see examples).

Portions and fragments of the proteoglycans of the invention are alsowithin the scope of the invention. A portion is typically at least five,10, 15, or 20 amino acids long. Preferred portions are those which aresufficient for exerting a biological activity, such as interacting witha DAPC component. Portions can comprise or consist of one or morespecific domain of a protein. Domains of biglycan and decorin includetwo cysteine-rich regions (included in the N- and C-terminal 40-50 aminoacids of mature biglycan) and leucine-rich repeats (LRRs). The “LRRregion” refers to the region of biglycan containing the repeats, andconsists essentially of amino acids 81-314. Each individual repeat isreferred to herein as an “LRR.” LRRs are believed to mediate protein:protein interactions and may thus be sufficient for stabilizing DAPCsand postsynaptic membranes. Based at least on the observation that bothdecorin and biglycan bind to MuSK and that the LLR region in both ofthese proteins is very similar, it is believed that the LRRs areinvolved in mediating the interaction of biglycan (and decorin) withMuSK and may be involved in mediating MuSK phosphorylation.

Another preferred biglycan of the invention consists of a portion ofbiglycan that is capable of binding to a sarcoglycan. It has been shownthat the α-sarcoglycan binding domain of human biglycan is located inthe N-terminal domain of the mature biglycan protein, i.e., amino acids38-80, and more specifically, amino acids 38-58 of SEQ ID NO: 9. The GAGchains are not necessary for binding to α-sarcgoglycan. It has also beenshown that the C-terminal cysteine-rich domain mediates interaction withγ-sarcoglycan. Accordingly, preferred biglycans of the invention includeportions of biglycan consisting of the N-terminal or the C-terminalcysteine-rich domain, i.e., amino acids 38-80 and 315-368 of SEQ ID NO:9. Combinations of certain domains of biglycan are also within the scopeof the invention.

Thus, preferred fragments consist of at least about 30 amino acids, atleast about 40 amino acids, 50, 60, 70, 80, 90, 100, 150, or 200 aminoacids. Short portions of the proteoglycans of the invention are termed“mini-proteoglycan of the invention.” For example, a biglycan corefragment of about 20, 30 or 40 amino acids is referred to as a“mini-biglycan.”

Human biglycan consists of 368 amino acids (SEQ ID NO: 9), of whichamino acids 1-19 constitute a signal peptide (GenBank Accession No.NP_(—)001702 and Fisher et al., supra). Thus biglycan without a signalpeptide consists of amino acids 20-368 of SEQ ID NO: 9. The maturebiglycan protein consists of amino acids 38-368 of SEQ ID NO: 9, sinceamino acids 1-37, being a pre-propeptide, are cleaved during processing.Amino acids 38-80 correspond to the N-terminal cysteine-rich region.About amino acids 81-314 corresponds to the leucine rich repeat region,containing 10 repeats of about 24 or 23 amino acids. The open readingframe in the cDNA encoding human biglycan corresponds to nucleotides121-1227 of SEQ ID NO: 7 and is represented as SEQ ID NO: 8. Thenucleotide sequence encoding a mature form of biglycan consists innucleotides 232-1227 of SEQ ID NO: 7.

In addition to agonists, the invention also provides antagonists ofbiglycan. An antagonist can be, e.g., a portion of the wild typeproteoglycan of the invention which inhibits the action of the wild typeproteoglycan, such as by competitively inhibiting the binding of thewild type proteoglycan to a target protein such as a component of aDAPC. Thus, an antagonist can be a dominant negative mutant.

The proteoglycan can be a mature form of the proteoglycan core, i.e.,deprived of the signal peptide, or the full length proteoglycan with thesignal peptide.

Preferred proteoglycans of the invention are encoded by nucleotidesequences which are at least about 70%, preferably at least about 80%,even more preferably at least about 85%, at least about 90%, at leastabout 95%, at least about 98%, and even more preferably at least about99% identical to the nucleotide sequence of an SLRP, e.g., biglycan, orortholog thereof, or portion thereof.

Preferred nucleic acids of the invention include those encoding apolypeptide comprising an amino acid sequence which is at least about70%, preferably at least about 80%, even more preferably at least about85%, at least about 90%, at least about 95%, at least about 98%, andeven more preferably at least about 99% identical to the nucleotidesequence of an SLRP, e.g., biglycan (e.g., SEQ ID NO: 7 or 8 encodinghuman biglycan) or DAG-125 or ortholog thereof, portion thereof. In oneembodiment, the nucleic acid encodes a polypeptide containing one ormore of SEQ ID NOs: 1-3 or SEQ ID NOs: 4-6 or 9.

Another aspect of the invention provides a nucleic acid which hybridizesunder stringent conditions to a nucleic acid encoding biglycan, e.g.,having one or more of SEQ ID NOS: 1 to 6 or 9, or complement thereof.Appropriate stringency conditions which promote DNA hybridization, forexample, 6.0× sodium chloride/sodium citrate (SSC) at about 45° C.,followed by a wash of 2.0×SSC at 50° C., are known to those skilled inthe art or can be found in Current Protocols in Molecular Biology, JohnWiley & Sons, N.Y. (1989), 6.3.1-6.3.6. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature of salt concentration may be held constant whilethe other variable is changed. In a preferred embodiment, a nucleic acidof the present invention will bind to one of SEQ ID NOS 1 to 6 orcomplement thereof or nucleic acid encoding a SLRP under moderatelystringent conditions, for example at about 2.0×SSC and about 40° C. In aparticularly preferred embodiment, a nucleic acid of the presentinvention will hybridize to a nucleotide sequence encoding one of SEQ IDNOS: 1 to 6 or 9, such as a nucleic acid having SEQ ID NO: 7 or 8, or acomplement thereof under high stringency conditions.

Methods for preparing compounds of the invention are well known in theart. For a compound of the invention which is a protein or a derivativethereof, the compound can be isolated from a tissue or the compound canbe recombinantly or synthetically produced. Isolation of the proteinfrom a tissue is described in the Examples. The proteins orproteoglycans of the invention isolated from tissue are preferably atleast about 70%, preferably at least about 80%, at least about 85%, atleast about 90%, at least about 95%, at least about 98% and mostpreferably, at least about 99% pure. Accordingly, preferred compoundscontain less than about 1%, and even more preferably less than about0.1% of material from which the compound was extracted.

The protein of the invention can also be produced recombinantly,according to methods well known in the art. Typically, a gene encodingthe protein is inserted into a plasmid or vector, and the resultingconstruct is then transfected into appropriate cells, in which theprotein is then expressed, and from which the protein is ultimatelypurified.

Accordingly, the present invention further pertains to methods ofproducing the subject proteins. For example, a host cell transfectedwith an expression vector encoding a protein of interest can be culturedunder appropriate conditions to allow expression of the protein tooccur. The protein may be secreted, by inclusion of a secretion signalsequence, and isolated from a mixture of cells and medium containing theprotein. Alternatively, the protein may be retained cytoplasmically andthe cells harvested, lysed and the protein isolated. A cell cultureincludes host cells, media and other byproducts. Suitable media for cellculture are well known in the art. The proteins can be isolated fromcell culture medium, host cells, or both using techniques known in theart for purifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the protein.

Thus, a coding sequence for a protein of the present invention can beused to produce a recombinant form of the protein via microbial oreukaryotic cellular processes. Ligating the polynucleotide sequence intoa gene construct, such as an expression vector, and transforming ortransfecting into hosts, either eukaryotic (yeast, avian, insect ormammalian) or prokaryotic (bacterial cells), are standard procedures.

Expression vehicles for production of a recombinant protein includeplasmids and other vectors. For instance, suitable vectors for theexpression of the instant fusion proteins include plasmids of the types:pBR322-derived plasmids, pEMBL-derived plasmids, pEX-derived plasmids,pBTac-derived plasmids and pUC-derived plasmids for expression inprokaryotic cells, such as E. coli.

A number of vectors exist for the expression of recombinant proteins inyeast. For instance, YEP24, YIPS, YEP51, YEP52, pYES2, and YRP17 arecloning and expression vehicles useful in the introduction of geneticconstructs into S. cerevisiae (see, for example, Broach et al., (1983)in Experimental Manipulation of Gene Expression, ed. M. Inouye AcademicPress, p. 83, incorporated by reference herein). These vectors canreplicate in E. coli due the presence of the pBR322 ori, and in S.cerevisiae due to the replication determinant of the yeast 2 micronplasmid. In addition, drug resistance markers such as ampicillin can beused.

The protein can be produced either in eukaryotic cells, e.g., mammaliancells, yeast cells, insect cell (baculovirus system) or in prokaryoticcells. However, if the protein is a proteoglycan, it is preferably toexpress it in a cell of the same type as that which normally producesthat particular proteoglycan. This assures that the correct types ofglucose side chain(s) are attached to the core (i.e., protein) of theproteoglycan. In particular, when biglycan is used in the invention, itis preferable that biglycan contains the appropriate GAG side chains.For example, when biglycan is used in the context of muscle cells, it ispreferable to produce biglycan in muscle cells, e.g., C2 muscle cells.The biglycan can also be produced in Torpedo cells, e.g., cells from theelectric organ of Torpedo.

Cells that can be used for producing a compound of the invention, e.g.,a proteoglycan can further be modified to increase the level and/oractivity of an enzyme that catalyzes posttranslational modifications,e.g., glycosylations or sulfonations. For example, a cell can betransformed or cotransfected with an expression construct encoding asulfotransferase, e.g., a chondroitin sulfotransferase, e.g., achondroitin-6-sulfotransferase (C6ST; Fukuta et al. (1995) J. Biol.Chem. 270: 18575), or a nervous system involved sulfotransferase(NSIST), described in Nastuk et al. (1998) J. Neuroscience 18: 7167.

Alternatively, a protein core of a proteoglycan can be produced in aprokaryote, which results in a protein without glucose side chains, andthe appropriate side chains can be added later, such as by syntheticchemistry. In yet another embodiment, a proteoglycan is produced in onetype of eukaryotic cell and the protein can be stripped of its sidechains, prior to adding the appropriate side chains. Methods forsynthetically adding glycan side chains to a protein are known in theart.

In a preferred embodiment, a recombinant protein of the invention, suchas biglycan or decorin, is produced using a vaccinia-based system, asdescribed in Krishnan et al. (1999) J. Biol. Chem. 294: 10945 and inHocking et al. (1996) J. Biol. Chem. 271:19571. Infection of musclecells with this vector encoding biglycan or decorin for example, resultsin the production of biglycan or decorin having muscle specific GAGchains. Biophysical studies, such as far UV circular dichroism showedthat these recombinant proteins retain their native structure. In aneven more preferred embodiment, these recombinant proteins areepitope-tagged, as further described herein, which facilitatesco-immunoprecipitation and binding studies.

For example, a proteoglycan of the invention can be produced in aeukaryotic cell using the vaccinia virus/T7 bacteriophage expressionsystem. A recombinant vaccinia virus, vBGN4 encoding the proteoglycan ofthe invention, e.g., mature biglycan protein, can be expressed as apolyhistidine fusion protein under control of the T7 phage promoter andexpressed, e.g., in HT-1080 cells and UMR106 cells, as described inHocking et al. (1996) J Biol Chem 271: 19571-7.

Immortalized cell lines, e.g., muscle cell lines, such as biglycannegative cell lines, can be obtained as described in Jat et al., PNAS(1991) 88: 5096-100; Noble et al., (1992) Brain Pathology 2: 39-46. Inone embodiment, a H-2K^(b)/tsA58 transgenic mouse is used. This mouse isa heterozygote harboring a thermolabile immortalizing gene (the tsA58mutant of SV40 large T antigen) under the control of aninterferon-inducible promoter (this mouse is available at CharlesRiver). When cells containing this gene are cultured, they proliferateindefinitely at 33° C. in the presence of interferon. However, when thetemperature is raised to 39° C. (at which temperature the tsA58 antigenis non-functional) and interferon is removed, the cells cease dividing.

This method has been used for growing a wide variety of cell types,including astrocytes, osteoclasts, trabecular network, and colonepithelial cells (Chambers et al., (1993) PNAS 90: 5578-82; Groves etal., (1993) Dev. Biol. 159: 87-104; Whitehead et al., (1993) PNAS 90:587-91; Noble et al., (1995) Transgenic Res. 4: 215-25; Tamm et al.,(1999) Invest. Ophtamol. Vis. Sci. 40: 1392-403. This technique is wellsuited for the production of muscle cell lines. For example, in onestudy alone 65 separate muscle cell lines were derived from animalsranging in age from neonates to four weeks (Morgan et al., (1994) Dev.Biol. 162 486-98). These lines were maintained for upwards of 80generations. Remarkably, they not only formed myotubes when shifted tonon-permissive conditions in culture, but also formed muscle whenimplanted into host mice. The H-2K^(b)/tsA58 transgenic method was alsoused by D. Glass and colleagues to produce a MuSK^(−/−) muscle cell line(Sugiyama et al., (1997) J. Cell Biol. 139: 181-91).

To produce conditionally immortalized cell lines, mice having a specificmutation, e.g., a deficiency in biglycan or MuSK, can be crossed withheterozygote H-2K^(b)/tsA58 transgenic mice. The crosses arestraightforward since only one copy of the gene is required for fullactivity. Muscle cells from neonatal animals can then be plated out andgrown under permissive conditions (33° C. with interferon).Proliferating cells can then be cloned and samples from each lineshifted to the non-permissive temperature and tested for their abilityto form myotubes. Wild type; decorin^(−/−); biglycan^(−/o); anddecorin^(−/−) biglycan^(−/o) cell lines are examples of cell lines whichcan be obtained using this technique.

In a further embodiment, the compound of the invention is a glycan orpolyssacharide. In fact, in certain applications, it may be that incertain cases, the core of a proteoglycan may not be necessary for thedesired activity, such as for stabilizing the DAPC by contacting one ormore components thereof. For example, it has been shown herein that theGAG side chains of biglycan are necessary for its interaction withα-dystroglycan, indicating that the interaction is likely to be mediatedby the GAG side chains.

The compounds of the invention can also be peptidomimetics or smallorganic molecules, which can be prepared, e.g., based on the structureof the proteoglyan.

Although the preferred method for treating subjects with a biglycan isby administration of the biglycan to the subject (based at least on theefficiency of biglycan when added to cell cultures, as described in theExamples), the proteoglycans of the invention can also be produced in asubject, by gene therapy techniques. Thus, e.g., a subject can receivean injection in a muscle (e.g., where the subject has a muscledystrophy) of a vector encoding a protein or proteoglycan of theinvention, such that the vector is capable of entering muscle cells andbeing expressed therein. Alternatively, the vector can be a viralvector, which is provided with the viral capside and the virus infectsthe cells, e.g., muscle cells and thereby deliver the vector. Methodsand vectors for gene therapy are well known in the art. Illustrativemethods are set forth below.

The preferred mammalian expression vectors contain both prokaryoticsequences to facilitate the propagation of the vector in bacteria, andone or more eukaryotic transcription units that are expressed ineukaryotic cells. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo,pSV2-dhfr, pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectorsare examples of mammalian expression vectors suitable for transfectionof eukaryotic cells. Some of these vectors are modified with sequencesfrom bacterial plasmids, such as pBR322, to facilitate replication anddrug resistance selection in both prokaryotic and eukaryotic cells.Alternatively, derivatives of viruses such as the bovine papilloma virus(BPV-1), or Epstein-Barr virus (pHEBo, pREP-derived and p205) can beused for transient expression of proteins in eukaryotic cells. Examplesof other viral (including retroviral) expression systems can be foundbelow in the description of gene therapy delivery systems. The variousmethods employed in the preparation of the plasmids and transformationof host organisms are well known in the art. For other suitableexpression systems for both prokaryotic and eukaryotic cells, as well asgeneral recombinant procedures, see Molecular Cloning: A LaboratoryManual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis (Cold SpringHarbor Laboratory Press, 1989) Chapters 16 and 17. In some instances, itmay be desirable to express the recombinant fusion proteins by the useof a baculovirus expression system. Examples of such baculovirusexpression systems include pVL-derived vectors (such as pVL1392, pVL1393and pVL941), pAcUW-derived vectors (such as pAcUW1), andpBlueBac-derived vectors (such as the -gal containing pBlueBac III).

In yet other embodiments, the subject expression constructs are derivedby insertion of the subject gene into viral vectors includingrecombinant retroviruses, adenovirus, adeno-associated virus, and herpessimplex virus-1, or recombinant bacterial or eukaryotic plasmids. Asdescribed in greater detail below, such embodiments of the subjectexpression constructs are specifically contemplated for use in variousin vivo and ex vivo gene therapy protocols.

Retrovirus vectors and adeno-associated virus vectors are generallyunderstood to be the recombinant gene delivery system of choice for thetransfer of exogenous genes in vivo, particularly into humans. Thesevectors provide efficient delivery of genes into cells, and thetransferred nucleic acids are stably integrated into the chromosomal DNAof the host. A major prerequisite for the use of retroviruses is toensure the safety of their use, particularly with regard to thepossibility of the spread of wild-type virus in the cell population. Thedevelopment of specialized cell lines (termed “packaging cells”) whichproduce only replication-defective retroviruses has increased theutility of retroviruses for gene therapy, and defective retroviruses arewell characterized for use in gene transfer for gene therapy purposes(for a review see Miller, A. D. (1990) Blood 76:271). Thus, recombinantretrovirus can be constructed in which part of the retroviral codingsequence (gag, pol, env) has been replaced by nucleic acid encoding afusion protein of the present invention rendering the retrovirusreplication defective. The replication defective retrovirus is thenpackaged into virions which can be used to infect a target cell throughthe use of a helper virus by standard techniques. Protocols forproducing recombinant retroviruses and for infecting cells in vitro orin vivo with such viruses can be found in Current Protocols in MolecularBiology, Ausubel, F. M. et al., (eds.) Greene Publishing Associates,(1989), Sections 9.10-9.14 and other standard laboratory manuals.Examples of suitable retroviruses include pLJ, pZIP, pWE and pEM whichare well known to those skilled in the art. Examples of suitablepackaging virus lines for preparing both ecotropic and amphotropicretroviral systems include SYMBOL 121 \f “Symbol”Crip, SYMBOL 121 \f“Symbol” Cre, SYMBOL 121 \f “Symbol” 2 and SYMBOL 121 \f “Symbol” Am.Retroviruses have been used to introduce a variety of genes into manydifferent cell types, including neural cells, epithelial cells,endothelial cells, lymphocytes, myoblasts, hepatocytes, bone marrowcells, in vitro and/or in vivo (see for example Eglitis et al., (1985)Science 230:1395-1398; Danos and Mulligan, (1988) PNAS USA 85:6460-6464;Wilson et al., (1988) PNAS USA 85:3014-3018; Armentano et al., (1990)PNAS USA 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039-8043; Ferryet al., (1991) PNAS USA 88:8377-8381; Chowdhury et al., (1991) Science254:1802-1805; van Beusechem et al., (1992) PNAS USA 89:7640-7644; Kayet al., (1992) Human Gene Therapy 3:641-647; Dai et al., (1992) PNAS USA89:10892-10895; Hwu et al., (1993) J. Immunol. 150:4104-4115; U.S. Pat.No. 4,868,116; U.S. Pat. No. 4,980,286; PCT Application WO 89/07136; PCTApplication WO 89/02468; PCT Application WO 89/05345; and PCTApplication WO 92/07573).

Furthermore, it has been shown that it is possible to limit theinfection spectrum of retroviruses and consequently of retroviral-basedvectors, by modifying the viral packaging proteins on the surface of theviral particle (see, for example PCT publications WO93/25234,WO94/06920, and WO94/11524). For instance, strategies for themodification of the infection spectrum of retroviral vectors include:coupling antibodies specific for cell surface antigens to the viral envprotein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan et al., (1992)J. Gen Virol 73:3251-3255; and Goud et al., (1983) Virology163:251-254); or coupling cell surface ligands to the viral env proteins(Neda et al., (1991) J. Biol. Chem. 266:14143-14146). Coupling can be inthe form of the chemical cross-linking with a protein or other variety(e.g. lactose to convert the env protein to an asialoglycoprotein), aswell as by generating fusion proteins (e.g. single-chain antibody/envfusion proteins). This technique, while useful to limit or otherwisedirect the infection to certain tissue types, and can also be used toconvert an ecotropic vector in to an amphotropic vector.

Another viral gene delivery system useful in the present inventionutilizes adenovirus-derived vectors. The genome of an adenovirus can bemanipulated such that it encodes a gene product of interest, but isinactivate in terms of its ability to replicate in a normal lytic virallife cycle (see, for example, Berkner et al., (1988) BioTechniques6:616; Rosenfeld et al., (1991) Science 252:431-434; and Rosenfeld etal., (1992) Cell 68:143-155). Suitable adenoviral vectors derived fromthe adenovirus strain Ad type 5 dl324 or other strains of adenovirus(e.g., Ad2, Ad3, Ad7 etc.) are well known to those skilled in the art.Recombinant adenoviruses can be advantageous in certain circumstances inthat they are not capable of infecting nondividing cells and can be usedto infect a wide variety of cell types, including airway epithelium(Rosenfeld et al., (1992) cited supra), endothelial cells (Lemarchand etal., (1992) PNAS USA 89:6482-6486), hepatocytes (Herz and Gerard, (1993)PNAS USA 90:2812-2816) and muscle cells (Quantin et al., (1992) PNAS USA89:2581-2584). Furthermore, the virus particle is relatively stable andamenable to purification and concentration, and as above, can bemodified so as to affect the spectrum of infectivity. Additionally,introduced adenoviral DNA (and foreign DNA contained therein) is notintegrated into the genome of a host cell but remains episomal, therebyavoiding potential problems that can occur as a result of insertionalmutagenesis in situations where introduced DNA becomes integrated intothe host genome (e.g., retroviral DNA). Moreover, the carrying capacityof the adenoviral genome for foreign DNA is large (up to 8 kilobases)relative to other gene delivery vectors (Berkner et al., supra;Haj-Ahmand and Graham (1986) J. Virol. 57:267). Mostreplication-defective adenoviral vectors currently in use and thereforefavored by the present invention are deleted for all or parts of theviral E1 and E3 genes but retain as much as 80% of the adenoviralgenetic material (see, e.g., Jones et al., (1979) Cell 16:683; Berkneret al., supra; and Graham et al., in Methods in Molecular Biology, E. J.Murray, Ed. (Humana, Clifton, N.J., 1991) vol. 7. pp. 109-127).Expression of the inserted chimeric gene can be under control of, forexample, the E1A promoter, the major late promoter (MLP) and associatedleader sequences, the viral E3 promoter, or exogenously added promotersequences.

Yet another viral vector system useful for delivery of the subjectchimeric genes is the adeno-associated virus (AAV). Adeno-associatedvirus is a naturally occurring defective virus that requires anothervirus, such as an adenovirus or a herpes virus, as a helper virus forefficient replication and a productive life cycle. (For a review, seeMuzyczka et al., Curr. Topics in Micro. and Immunol. (1992) 158:97-129).It is also one of the few viruses that may integrate its DNA intonon-dividing cells, and exhibits a high frequency of stable integration(see for example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol.7:349-356; Samulski et al., (1989) J. Virol. 63:3822-3828; andMcLaughlin et al., (1989) J. Virol. 62:1963-1973). Vectors containing aslittle as 300 base pairs of AAV can be packaged and can integrate. Spacefor exogenous DNA is limited to about 4.5 kb. An AAV vector such as thatdescribed in Tratschin et al., (1985) Mol. Cell. Biol. 5:3251-3260 canbe used to introduce DNA into cells. A variety of nucleic acids havebeen introduced into different cell types using AAV vectors (see forexample Hermonat et al., (1984) PNAS USA 81:6466-6470; Tratschin et al.,(1985) Mol. Cell. Biol. 4:2072-2081; Wondisford et al., (1988) Mol.Endocrinol. 2:32-39; Tratschin et al., (1984) J. Virol. 51:611-619; andFlotte et al., (1993) J. Biol. Chem. 268:3781-3790).

Other viral vector systems that may have application in gene therapyhave been derived from herpes virus, vaccinia virus, and several RNAviruses. In particular, herpes virus vectors may provide a uniquestrategy for persistence of the recombinant gene in cells of the centralnervous system and ocular tissue (Pepose et al., (1994) InvestOphthalmol V is Sci 35:2662-2666).

In addition to viral transfer methods, such as those illustrated above,non-viral methods can also be employed to cause expression of a proteinin the tissue of an animal. Most nonviral methods of gene transfer relyon normal mechanisms used by mammalian cells for the uptake andintracellular transport of macromolecules. In preferred embodiments,non-viral gene delivery systems of the present invention rely onendocytic pathways for the uptake of the gene by the targeted cell.Exemplary gene delivery systems of this type include liposomal derivedsystems, poly-lysine conjugates, and artificial viral envelopes.

In a representative embodiment, a gene encoding a protein of interestcan be entrapped in liposomes bearing positive charges on their surface(e.g., lipofectins) and (optionally) which are tagged with antibodiesagainst cell surface antigens of the target tissue (Mizuno et al.,(1992) No Shinkei Geka 20:547-551; PCT publication WO91/06309; Japanesepatent application 1047381; and European patent publication EP-A-43075).For example, lipofection of muscle, neural or cardiac cells can becarried out using liposomes tagged with monoclonal antibodies againstspecific tissue-associated antigens (Mizuno et al., (1992) Neurol. Med.Chir. 32:873-876).

In yet another illustrative embodiment, the gene delivery systemcomprises an antibody or cell surface ligand which is cross-linked witha gene binding agent such as poly-lysine (see, for example, PCTpublications WO93/04701, WO92/22635, WO92/20316, WO92/19749, andWO92/06180). For example, any of the subject gene constructs can be usedto transfect specific cells in vivo using a soluble polynucleotidecarrier comprising an antibody conjugated to a polycation, e.g.poly-lysine (see U.S. Pat. No. 5,166,320). It will also be appreciatedthat effective delivery of the subject nucleic acid constructs via-mediated endocytosis can be improved using agents which enhance escapeof the gene from the endosomal structures. For instance, wholeadenovirus or fusogenic peptides of the influenza HA gene product can beused as part of the delivery system to induce efficient disruption ofDNA-containing endosomes (Mulligan et al., (1993) Science 260-926;Wagner et al., (1992) PNAS USA 89:7934; and Christiano et al., (1993)PNAS USA 90:2122).

Nucleic acids encoding biglycan proteins can also be administered to asubject as “naked” DNA, as described, e.g., in U.S. Pat. No. 5,679,647and related patents by Carson et al., in WO 90/11092 and Felgner et al.(1990) Science 247: 1465.

In clinical settings, the gene delivery systems can be introduced into apatient by any of a number of methods, each of which is familiar in theart. For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g. by intravenous injection,and specific transduction of the construct in the target cells occurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of the gene,or a combination thereof. In other embodiments, initial delivery of therecombinant gene is more limited with introduction into the animal beingquite localized. For example, the gene delivery vehicle can beintroduced by catheter (see U.S. Pat. No. 5,328,470) or by stereotacticinjection (e.g. Chen et al., (1994) PNAS USA 91: 3054-3057).

The gene encoding the proteoglycan of the invention can be under thecontrol of a constitutive, or inducible promoter. These are well knownin the art.

Methods for determining whether a compound has a biological activity ofa biglycan protein are described in the Examples. A biological activityof a biglycan protein is intended to refer to one or more of: theability to maintain the integrity of a plasma membrane; the ability tostabilize DAPCs on plasma membranes; the ability to bind to one or morecomponents of DAPCs; e.g., binding to α-dystroglycan, binding to asarcoglycan component, such as α-sarcoglycan; phosphorylation ofα-sarcoglycan; binding to MuSK; stimulating the formation ofneuromuscular junctions, such as by stimulating postsynapticdifferentiation; stimulating AChR aggregation; stimulation of MuSKphosphorylation and potentiation of agrin-induced MuSK phosphorylation.Such methods can further be adapted for screening libraries of compoundsfor identifying compounds having one or more of the above-describedactivities.

Breakdown of cytoplasmic membranes, e.g., the presence of “leakymembranes” can be determined by assays which measure the release ofcreatine kinase or the absorption of Evans Blue dye, as described, e.g.,in Tinsley et al. (1996) Nature 384: 349 and Straub et al. (1997) J.Cell Biol. 139: 375).

The compounds of the invention can also be tested in a variety of animalmodels, in particular the mdx mice, which are dystrophin negative (seeExamples).

IV. Methods of Treatment

The invention provides therapeutic and prophylactic methods of treatmentof disorders including muscular, neuromuscular, and neurologicaldisorders. Therapeutic methods are intended to eliminate or at leastreduce at least one symptom of a disease or disorder, and preferablycure the disease or disorder. Prophylactic methods include thoseintended to prevent the appearance of a disease or disorder, i.e., amethod which is intended to combat the appearance of the disease ordisorder.

As described herein, biglycan was shown to bind to α-dystroglycan and tosarocoglycans, and thereby functions as a link between variouscomponents of DAPCs. Furthermore, biglycan levels were found to be highin muscle cells of mice lacking dystrophin (mdx mice, which are a modelof muscular dystrophy). Since the absence of dystrophin in muscle cellsis known to destabilize the cytoplasmic membrane, the upregulation ofbiglycan in dystrophin negative muscle cells may be a compensatorymechanism for the absence of dystrophin. Accordingly, the inventionprovides for methods for preventing and treating diseases or disordersthat are associated with plasma membrane instability or organization, inparticular, an instability resulting from an abnormal DAPC on the plasmamembrane. Since the DAPC is found on the membrane of muscle cells,diseases that can be treated according to the invention include diseasesof the muscle, such as muscular dystrophies and muscle atrophy.

In that regard, one promising path for treatment and potentially a curefor muscular dystrophy the activation of an endogenous compensatorymechanism based upon the regulated expression of utrophin. Utrophin is ahomolog of dystrophin which shares numerous structural and functionalproperties with it. However, in both normal and in Duchenne's muscleutrophin is only expressed at a fraction of the muscle membrane: theneuromuscular junction and the myotendinous junction. The bulk of themembrane has no utrophin. However, in animal models it has been shownthat forced expression of utrophin in muscle lacking dystrophin leads torestoration of the DAPC in the muscle membrane and to rescue of thedystrophic phenotype. Since the utrophin gene is normal in Duchennepatients, a method to activate its expression in muscle and/or to targetit to the muscle membrane could serve to restore the DAPC to themembrane and thus promote the health of the muscle cells.

Several lines of evidence, many of them arising from observations madeby the inventors indicate that the small leucine-rich repeatproteoglycan biglycan could be a method for regulating utrophinexpression and localization. It has been demonstrated that the proteinagrin can cause an upregulation of utrophin expression and direct it tobe localized to specific domains on the cell surface. The signalingreceptor for agrin is the receptor tyrosine kinase MuSK. It has beenobserved that agrin can also induce the tyrosine phosphorylation of α-and γ-sarcoglycan in cultured myotubes. It was also observed thatbiglycan can also regulate the tyrosine phosphorylation of α- andγ-sarcoglycan. Moreover, the receptor tyrosine kinase MuSK is requiredfor this biglycan-induced tyrosine phosphorylation of these proteins.Further, biglycan can bind to MuSK. These observations indicate thatbiglycan can act directly to organize the DAPC, including utrophin, onthe muscle cell surface.

Thus the present invention contemplates the treatment of these disorderswith biglycan therapeutics which upregulate utrophin, activate MuSKand/or induce phosphorylation of sarcoglycans.

Merely to illustrate, biglycan polypeptides, peptides or peptidomimeticscan be delivered to patients with muscular dystrophy or other conditionswhere muscle atrophies to upregulate the endogenous utrophin geneexpression and/or to promote the localization of utrophin to the musclemembrane. In such embodiments, the biglycan polypeptide may be deliveredin the form of a polypeptide in and of itself, or as part of a fusionprotein, e.g., fused to a humanized antibody sequence or similar carrierentity. Biglycan polypeptides can be delivered by nucleic acid-basedmethods including as plasmid DNA, in viral vectors, or other modalitieswhere the nucleic acid sequences encoding biglycan are introduced intopatients. The delivery of a biglycan therapeutic can serve to heal themuscle fibers from within by directing the increased expression andregulated localization of utrophin to the muscle cell surface withconcomitant restoration of the remainder of the dystrophin-associatedprotein complex.

However, the present invention also contemplates the use of agents whichact upstream of biglycan, e.g., which induce the expression of nativebiglycan genes. Treatment with such agents as angotensin II, sodiumsalicylate, forskolin and 8-bromo-cAMP, for example, results insignificant increases in expression of biglycan and can be used as partof a treatment protocol for such disorders.

Furthermore, since DAPCs are also found on other cell types, theinvention also provides methods for treating diseases associated withany abnormal DAPC. For example, DAPC are present in the brain, andsince, in addition, agrin has been found in senile plaques in patientswith Alzheimers's disease, neurological diseases can also be treated orprevented according to the methods of the invention. A furtherindication that neurological disorders can be treated or preventedaccording to the methods described herein is based on the observationthat patients with muscular dystrophy often also suffer from peripheraland central nervous system disorder. Accordingly, about one third ofpatients with Duchenne Muscular Dystrophy have a mental affliction, inparticular, mental retardation. Thus, dystrophin, and hence, DAPCs, arebelieved to play a role in the nervous system.

Patients with Duchenne's Muscular Dystrophy also have diaphragmproblems, indicating a role for dystrophin, and possibly DAPCs indiaphragms. Thus, therapeutics of the invention would also find anapplication in disorders associated with diaphragm abnormalities.

It should be noted that diseases that can be treated or preventedinclude not only those in which biglycan is abnormal, but more generallyany disease or condition that is associated with a defect that can beimproved or cured by biglycan. In particular, diseases that arecharacterized by a defect or an abnormality in any component of the DAPCor component associated therewith, thereby resulting, e.g., in anunstable plasma membrane, can be treated or prevented according to themethods of the invention, provided that the proteoglycan of theinvention can at least partially cure the defect resulting from thedeficient component. In particular, diseases that can be treatedaccording to the method of the invention include any disease associatedwith an unstable DAPC, which can be rendered more stable by the presenceof a proteoglycan of the invention.

Furthermore, since biglycan was shown to bind to, and phosphorylatesMuSK, a receptor which is known for mediating agrin-induced stimulationof neuromuscular junction formation, in particular postsynaptic membranedifferentiation, to potentiateagrin-induced AChR aggregation, and tocorrect a defective agrin-induced AChR aggregation in myotubes ofbiglycan negative mice by its addition to the myotubes, the inventionalso provides methods for preventing and treating diseases or disordersof neuromuscular junctions, such as neuromuscular disorders. Mostinterestingly, exogenously added biglycan was shown to be able tocorrect a defective agrin-induced AChR aggregation in myotubes ofbiglycan negative mice.

A. Exemplary Diseases and Disorders:

Diseases or disorders that are characterized by a destabilization orimproper organization of the plasma membrane of specific cell typesinclude muscular dystrophies (MDs), a group of genetic degenerativemyopathies characterized by weakness and muscle atrophy without nervoussystem involvement. The three main types are pseudohypertrophic(Duchenne, Becker), limb-girdle, and facioscapulohumeral. For example,muscular dystrophies and muscular atrophies are characterized by abreakdown of the muscle cell membrane, i.e., they are characterized byleaky membranes, which are believed to result from a mutation in acomponent of the DAPC., i.e., dystrophin. Mutations in the sarcoglycansare also known to result in muscular dystrophies and leaky membranes.Accordingly, the invention provides for methods for treating orpreventing diseases associated with mutations in dystrophin and/or insarcoglycans or other component of DAPCs, in particular musculardystrophies.

Dystrophin abnormalities are responsible for both the milder Becker'sMuscular Dystrophy (BMD) and the severe Duchenne's Muscular Dystrophy(DMD). In BMD dystrophin is made, but it is abnormal in either sizeand/or amount. The patient is mild to moderately weak. In DMD no proteinis made and the patient is wheelchair-bound by age 13 and usually diesby age 20.

Another type of dystrophy that can be treated according to the methodsof the invention includes congenital muscular dystrophy (CMD), a verydisabling muscle disease of early clinical onset, is the most frequentcause of severe neonatal hypotonia. Its manifestations are noticed atbirth or in the first months of life and consist of muscle hypotonia,often associated with delayed motor milestones, severe and earlycontractures and joint deformities. Serum creatine kinase is raised, upto 30 times the normal values, in the early stage of the disease, andthen rapidly decreases. The histological changes in the muscle biopsiesconsist of large variation in the size of muscle fibers, a few necroticand regenerating fibers, marked increase in endomysial collagen tissue,and no specific ultrastructural features. The diagnosis of CMD has beenbased on the clinical picture and the morphological changes in themuscle biopsy, but it cannot be made with certainty, as other muscledisorders may present with similar clinico-pathological features. Withinthe group of diseases classified as CMD, various forms have beenindividualized. The two more common forms are the occidental and theJapanese, the latter being associated with severe mental disturbances,and usually referred to as Fukuyama congenital muscular dystrophy(FCMD).

One form of congenital muscular dystrophy (CMD) has recently beencharacterized as being caused by mutations in the laminin alpha 2-chaingene. Laminin is a protein that associates with DAPCs. Thus, theinvention also provides methods for treating diseases that areassociated with abnormal molecules which normally associate with DAPCs.

Other muscular dystrophies within the scope of the invention includelimb-girdle muscular dystrophy (LGMD), which represents a clinically andgenetically heterogeneous class of disorders. These dystrophies areinherited as either autosomal dominant or recessive traits. An autosomaldominant form, LGMD1A, was mapped to 5q31-q33 (Speer, M. C. et al., Am.J. Hum. Genet. 50:1211, 1992; Yamaoka, L. Y. et al., Neuromusc. Disord.4:471, 1994), while six genes involved in the autosomal recessive formswere mapped to 15q15.1 (LGMD2A)(Beckmann, J. S. et al., C. R. Acad. Sci.Paris 312:141, 1991), 2p16-p13 (LGMD2B)(Bashir, R. et al., Hum. Mol.Genet. 3:455, 1994), 13q12 (LGMD2C)(Ben Othmane, K. et al., NatureGenet. 2:315, 1992; Azibi, K. et al., Hum. Mol. Genet. 2:1423, 1993),17q12-q21.33 (LGMD2D)(Roberds, S. L. et al., Cell 78:625, 1994; McNally,E. M., et. al., Proc. Nat. Acad. Sci. U.S.A. 91:9690, 1994), 4q12(LG1MD2E)(Lim, L. E., et. al., Nat. Genet. 11:257, 1994; Bonnemann, C.G. et al. Nat. Genet. 11:266, 1995), and most recently to 5q33-q34(LGMD2F) (Passos-Bueno, M. R., et. al., Hum. Mol. Genet. 5:815, 1996).Patients with LGMD2C, 2D and 2E have a deficiency of components of thesarcoglycan complex resulting from mutations in the genes encodinggamma-, alpha-, and beta-sarcoglycan, respectively. The gene responsiblefor LGMD2A has been identified as the muscle-specific calpain, whereasthe genes responsible for LGMD1A, 2B and 2F are still unknown.

Yet other types of muscular dystrophies that can be treated according tothe methods of the invention include Welander distal myopathy (WDM),which is an autosomal dominant myopathy with late-adult onsetcharacterized by slow progression of distal muscle weakness. Thedisorder is considered a model disease for hereditary distal myopathies.The disease is linked to chromosome 2p13. Another muscular dystrophy isMiyoshi myopathya, which is a distal muscular dystrophy that is causedby mutations in the recently cloned gene dysferlin, gene symbol DYSF(Weiler et al. (1999) Hum Mol Genet. 8: 871-7). Yet other dystrophiesinclude Hereditary Distal Myopathy, Benign Congenital Hypotonia, CentralCore disease, Nemaline Myopathy, and Myotubular (centronuclear)myopathy.

Other diseases that can be treated or prevented according to the methodsof the invention include those characterized by tissue atrophy, e.g.,muscle atrophy, other than muscle atrophy resulting from musculardystrophies, provided that the atrophy is stopped or slowed down upontreatment with a therapeutic of the invention. Furthermore, theinvention also provides methods for reversing tissue atrophies, e.g.,muscle atrophies. This can be achieved, e.g., by providing to theatrophied tissue a therapeutic of the invention, such as DAG-125 ormammalian ortholog thereof, or biglycan.

Muscle atrophies can result from denervation (loss of contact by themuscle with its nerve) due to nerve trauma; degenerative, metabolic orinflammatory neuropathy (e.g., GuillianBarre syndrome), peripheralneuropathy, or damage to nerves caused by environmental toxins or drugs.In another embodiment, the muscle atrophy results from denervation dueto a motor neuronopathy. Such motor neuronopathies include, but are notlimited to: adult motor neuron disease, including Amyotrophic LateralSclerosis (ALS or Lou Gehrig's disease); infantile and juvenile spinalmuscular atrophies, and autoimmune motor neuropathy with multifocalconduction block. In another embodiment, the muscle atrophy results fromchronic disuse. Such disuse atrophy may stem from conditions including,but not limited to: paralysis due to stroke, spinal cord injury;skeletal immobilization due to trauma (such as fracture, sprain ordislocation) or prolonged bed rest. In yet another embodiment, themuscle atrophy results from metabolic stress or nutritionalinsufficiency, including, but not limited to, the cachexia of cancer andother chronic illnesses, fasting or rhabdomyolysis, endocrine disorderssuch as, but not limited to, disorders of the thyroid gland anddiabetes.

Since muscle tissue atrophy and necrosis are often accompanied byfibrosis of the affected tissue, the reversal or the inhibition ofatrophy or necrosis can also result in an inhibition or reversal offibrosis.

In addition, the therapeutics of the invention may be of use in thetreatment of acquired (toxic or inflammatory) myopathies. Myopathieswhich occur as a consequence of an inflammatory disease of muscle,include, but not limited to polymyositis and dermatomyositis. Toxicmyopathies may be due to agents, including, but are not limited toadiodarone, chloroquine, clofibrate, colchicine, doxorubicin, ethanol,hydroxychloroquine, organophosphates, perihexyline, and vincristine.

Neuromuscular dystrophies within the scope of the invention includemyotonic dystrophy. Myotonic dystrophy (DM; or Steinert's disease) is anautosomal dominant neuromuscular disease which is the most common formof muscular dystrophy affecting adults. The clinical picture in DM iswell established but exceptionally variable (Harper, P. S., MyotonicDystrophy, 2nd ed., W. B. Saunders Co., London, 1989). Althoughgenerally considered a disease of muscle, with myotonia, progressiveweakness and wasting, DM is characterized by abnormalities in a varietyof other systems. DM patients often suffer from cardiac conductiondefects, smooth muscle involvement, hypersomnia, cataracts, abnormalglucose response, and, in males, premature balding and testicularatrophy (Harper, P. S., Myotonic Dystrophy, 2nd ed., W. B. Saunders Co.,London, 1989). The mildest form, which is occasionally difficult todiagnose, is seen in middle or old age and is characterized by cataractswith little or no muscle involvement. The classical form, showingmyotonia and muscle weakness, most frequently has onset in early adultlife and in adolescence. The most severe form, which occurscongenitally, is associated with generalized muscular hypoplasia, mentalretardation, and high neonatal mortality. This disease and the geneaffected is further described in U.S. Pat. No. 5,955,265.

Another neuromuscular disease is spinal muscular atrophy (“SMA”), whichis the second most common neuromuscular disease in children afterDuchenne muscular dystrophy. SMA refers to a debilitating neuromusculardisorder which primarily affects infants and young children. Thisdisorder is caused by degeneration of the lower motor neurons, alsoknown as the anterior horn cells of the spinal cord. Normal lower motorneurons stimulate muscles to contract. Neuronal degeneration reducesstimulation which causes muscle tissue to atrophy (see, e.g., U.S. Pat.No. 5,882,868).

The above-described muscular dystrophies and myopathies are skeletalmuscle disorders. However, the invention also pertains to disorders ofsmooth muscles, e.g., cardiac myopathies, including hypertrophiccardiomyopathy, dilated cardiomyopathy and restrictive cardiomyopathy.At least certain smooth muscles, e.g., cardiac muscle, are rich insarcoglycans. Mutations in sarcoglycans can result in sarcolemmalinstability at the myocardial level (see, e.g., Melacini (1999) MuscleNerve 22: 473). For example, animal models in which a sarcoglycan ismutated show cardiac creatine kinase elevation. In particular, it hasbeen shown that delta-sarcoglycan (Sgcd) null mice developcardiomyopathy with focal areas of necrosis as the histological hallmarkin cardiac and skeletal muscle. The animals also showed an absence ofthe sarcoglycan-sarcospan (SG-SSPN) complex in skeletal and cardiacmembranes. Loss of vascular smooth muscle SG-SSPN complex was associatedwith irregularities of the coronary vasculature. Thus, disruption of theSG-SSPN complex in vascular smooth muscle perturbs vascular function,which initiates cardiomyopathy and exacerbates muscular dystrophy(Coral-Vazquez et al. (1999) Cell 98: 465).

Similarly to delta-sarcoglycan negative mice, mice lacking γ-sarcoglycanshowed pronounced dystrophic muscle changes in early life (Hack et al.(1998) J Cell Biol 142: 1279). By 20 wk of age, these mice developedcardiomyopathy and died prematurely. Furthermore, apoptotic myonucleiwere abundant in skeletal muscle lacking γ-sarcoglycan, suggesting thatprogrammed cell death contributes to myofiber degeneration. Vitalstaining with Evans blue dye revealed that muscle lacking γ-sarcoglycandeveloped membrane disruptions like those seen in dystrophin-deficientmuscle. It was also shown that the loss of γ-sarcoglycan producedsecondary reduction of beta- and delta-sarcoglycan with partialretention of α- and epsilon-sarcoglycan, indicating that beta-, γ-, anddelta-sarcoglycan function as a unit. Since the other components of thecytoplasmic membrane complex were functional, the complex could bestabilized by the presence of a therapeutic of the invention.

In addition to animal models, certain cardiomyopathies in humans havebeen linked to mutations in dystrophin, dystroglycans or sarcoglycans.For example, dystrophin has been identified as the gene responsible forX-linked dilated cardiomyopathy (Towbin J. A. (1998) Curr Opin Cell Biol10: 131, and references therein). In this case, the dystrophin genecontained a 5′-mutation which results in cardiomyopathy withoutclinically-apparent skeletal myopathy (Bies et al. (1997) J Mol CellCardiol 29: 3175.

Furthermore, cardiomyopathy was also found in subjects having Duchenne'sMuscular Dystrophy (associated with a mutated dystrophin), or othertypes of muscular dystrophies, such as Limb Girdle Muscular Dystrophy.For example, dilated cardiomyopathy was present in one autosomaldominant case and in three advanced autosomal recessive or sporadicpatients, of whom two were found to have alpha sarcoglycan deficiency.Two of these three patients and three other cases showed ECGabnormalities known to be characteristic of the dystrophinopathies. Astrong association between the absence of alpha sarcoglycan and thepresence of dilated cardiomyopathy was found. In six autosomal dominantcases there were atrioventricular (AV) conduction disturbances,increasing in severity with age and in concomitant presence of muscleweakness. Pacemaker implantation was necessary in certain of thesepatients (see van der Kooi (1998) Heart 79: 73).

Therapeutics of the invention can also be used to treat or preventcardiomyopathy, e.g., dilated cardiomyopathy, of viral origin, e.g.,resulting from an enterovirus infection, e.g., a Coxsackievirus B3. Ithas been shown that purified Coxsackievirus protease 2A cleavesdystrophin in vitro and during Coxsackievirus infection of culturedmyocytes and in infected mouse hearts, leading to impaired dystrophinfunction (Badorff et al. (1999) Nat Med 5: 320. Cleavage of dystrophinresults in disruption of the dystrophin-associated glycoproteinsα-sarcoglycan and beta-dystroglycan. Thus, cardiomyopathy could beprevented or reversed by administration of a therapeutic of theinvention to a subject having been infected with a virus causingcardiomyopathy, e.g., by disruption of dystrophin or a proteinassociated therewith. Administration of the therapeutic couldrestabilize or reorganize the cytoplasmic membrane of affected cardiaccells.

Thus, the therapeutics of the invention can also be used to prevent orto treat smooth muscle disorders, such as cardiac myopathies, and tostop atrophy and/or necrosis of cardiac smooth muscle tissue. Thetreatment can also be used to promote survival of myocytes.

Neurological disorders that can be treated according to the methods ofthe invention include polymyositis, and neurogenic disorders. Anotherneurological disease that can be treated is Alzheimers' disease.

Other diseases that can be treated according to the methods of theinvention include those in which the proteoglycan of the invention ispresent at abnormal levels, or has an abnormal activity, relative tothat in normal subjects. For example, a disease or disorder could becaused by a lower level of biglycan, resulting in, e.g., unstablecytoplasmic membranes. Alternatively, a disease or disorder could resultfrom an abnormally high level or activity of biglycan, resulting in,e.g., overstimulation of MuSK or over-aggregation of AChRs (see below).

Yet other diseases or disorders that are within the scope of theinvention include those that are associated with an abnormal interactionbetween a proteoglycan of the invention and another molecule (other thanthose of the DAPC or MuSK), e.g., a complement factor, such as C1q. Forexample, it has been shown that C1q interacts with biglycan (Hocking etal. (1996) J. Biol. Chem. 271: 19571). It is also known that binding ofC1q to cell surfaces mediates a number of biological activitiesincluding enhancement of phagocytosis and stimulation of superoxideproduction. Thus, since biglycan binds to C1q, biglycan or anotherproteoglycan or core thereof, of the invention could be used to inhibitthe binding of C1q to its receptor on cell surfaces to inhibit one ormore of such biological activities. In addition, compounds of theinvention which inhibit the interaction between C1q or other complementcomponent and a cell surface can also be used to inhibit complementmediated necrosis of the cells and tissues containing such cells.

Also within the scope of the invention are methods for preventing orinhibiting infections of cells by microorganisms, e.g., viruses. Forexample, it has been shown that dystroglycan is a receptor via whichcertain microorganisms enter eukaryotic cells (Science (1998) 282:2079). Thus, by administrating to a subject a therapeutic of theinvention which occupies the site on dystroglycan molecules to which themicroorganism binds, entering of the microorganism into the cell can beinhibited. This method can be used, e.g., to prevent or inhibit LassaFever virus and lymphocytic choriomeningitis virus (LCMV) infection, aswell as infection by other arenaviruses, including Oliveros, and Mobala.Soluble alphα-dystroglycan was shown to block both LCMV and LFVinfection (Science (1998) 282: 2079).

In addition to cell cultures, e.g., established from patients having,e.g., a muscular dystrophy, various animal models can be used to selectthe most appropriate therapeutic for treating a disease. In particular,to identify a therapeutic for use in preventing or treating a musculardystrophy or cardiomyophaty associated with a mutated or absent DAPCcomponent or, mice having mutated versions of these proteins, or havingnull mutations in the genes encoding these proteins, can be used. Forexample, mice having a disrupted sarcoglycan, such as delta-sarcoglycan,can be used. Such mice are described, e.g., Coral-Vazquez et al. (1999)Cell 98: 465. Alternatively, mice deficient in dystrophin (mdx mice), orin α- or γ-sarcoglycans can be used. Such mice have been describedherein and in the literature. Additional mice can be made according toknown methods in the art. In an illustrative embodiment to identifytherapeutics, different therapeutics are administered todelta-sarcoglycan null mice, and the effect of the therapeutics areevaluated by studying cardiac function. Another animal model that can beused for this purpose is the cardiomyopathic hamster that does notexpress delta-sarcoglycan due to a genomic deletion. This rat is ananimal model for autosomal recessive cardiomyopathy., and is furtherdescribed in Sakamoto et al. FEBS Lett 1999 (1999) 44: 124.

V. Effective Dose and Administration of Therapeutic Compositions

The above-described diseases or disorders can be treated or amelioratedin a subject by administering to the subject a pharmaceuticallyefficient amount of a bigylcan therapeutic of the invention. Dependingon whether the disease is caused by higher levels or activity or bylower levels or activity of biglycan, an agonist or an antagonistbiglycan therapeutic is administered to a subject having the disease.Although a person of skill in the art will be able to predict whichtherapeutic to administer for treating any of the diseases of theinvention, tests can be performed to determine the appropriatetherapeutic to administer. Such tests can use, e.g., animal models ofthe disease. Alternatively, in cases where diseases are due to amutation in, e.g., biglycan, in vitro tests can be undertaken todetermine the effect of the mutation. This will allow the determinationof what type of therapeutic should be administered to a subject havingthis type of mutation.

The bigylcan therapeutic can also be a compound which modulates, i.e.,inhibits or stimulates, expression of biglycan, or mammalian orthologthereof, or biglycan. Such compounds can be identified as furtherdescribed herein.

Another manner of administering a therapeutic of the invention to asubject is by preparing cells expressing and secreting the proteoglycanof interest, inserting the cells into a matrix and administering thismatrix to the subject at the desired location. Thus, cells engineered inaccordance with this invention may also be encapsulated, e.g. usingconventional biocompatible materials and methods, prior to implantationinto the host organism or patient for the production of a therapeuticprotein. See e.g. Hguyen et al, Tissue Implant Systems and Methods forSustaining viable High Cell Densities within a Host, U.S. Pat. No.5,314,471 (Baxter International, Inc.); Uludag and Sefton, 1993, JBiomed. Mater. Res. 27(10):1213-24 (HepG2 cells/hydroxyethylmethacrylate-methyl methacrylate membranes); Chang et al, 1993, Hum GeneTher 4(4):433-40 (mouse Ltk-cells expressing hGH/immunoprotectiveperm-selective alginate microcapsules; Reddy et al, 1993, J Infect Dis168(4):1082-3 (alginate); Tai and Sun, 1993, FASEB J 7(11):1061-9 (mousefibroblasts expressing hGH/alginate-poly-L-lysine-alginate membrane); Aoet al, 1995, Transplanataion Proc. 27(6):3349, 3350 (alginate); Rajotteet al, 1995, Transplantation Proc. 27(6):3389 (alginate); Lakey et al,1995, Transplantation Proc. 27(6):3266 (alginate); Korbutt et al, 1995,Transplantation Proc. 27(6):3212 (alginate); Dorian et al, U.S. Pat. No.5,429,821 (alginate); Emerich et al, 1993, Exp Neurol 122(1):37-47(polymer-encapsulated PC12 cells); Sagen et al, 1993, J Neurosci13(6):2415-23 (bovine chromaffin cells encapsulated in semipermeablepolymer membrane and implanted into rat spinal subarachnoid space);Aebischer et al, 1994, Exp Neurol 126(2):151-8 (polymer-encapsulated ratPC12 cells implanted into monkeys; see also Aebischer, WO 92/19595);Savelkoul et al, 1994, J Immunol Methods 170(2):185-96 (encapsulatedhybridomas producing antibodies; encapsulated transfected cell linesexpressing various cytokines); Winn et al, 1994, PNAS USA 91(6):2324-8(engineered BHK cells expressing human nerve growth factor encapsulatedin an immunoisolation polymeric device and transplanted into rats);Emerich et al, 1994, Prog Neuropsychopharmacol Biol Psychiatry18(5):935-46 (polymer-encapsulated PC12 cells implanted into rats);Kordower et al, 1994, PNAS USA 91(23):10898-902 (polymer-encapsulatedengineered BHK cells expressing hNGF implanted into monkeys) and Butleret al WO 95/04521 (encapsulated device). The cells may then beintroduced in encapsulated form into an animal host, preferably a mammaland more preferably a human subject in need thereof. Preferably theencapsulating material is semipermeable, permitting release into thehost of secreted proteins produced by the encapsulated cells. In manyembodiments the semipermeable encapsulation renders the encapsulatedcells immunologically isolated from the host organism in which theencapsulated cells are introduced. In those embodiments the cells to beencapsulated may express one or more proteoglycans of the host speciesand/or from viral proteins or proteins from species other than the hostspecies.

Alternatively, the therapeutic is a nucleic acid encoding the core of aproteoglycan of the invention. Thus, a subject in need thereof, mayreceive a dose of viral vector encoding the protein of interest, whichmay be specifically targeted to a specific tissue, e.g., a dystrophictissue. The vector can be administered in naked form, or it can beadministered as a viral particle (further described herein). For thispurpose, various techniques have been developed for modification oftarget tissue and cells in vivo. A number of viral vectors have beendeveloped, such as described above, which allow for transfection and, insome cases, integration of the virus into the host. See, for example,Dubensky et al. (1984) Proc. Natl. Acad. Sci. USA 81, 7529-7533; Kanedaet al., (1989) Science 243, 375-378; Hiebert et al. (1989) Proc. Natl.Acad. Sci. USA 86, 3594-3598; Hatzoglu et al. (1990) J. Biol. Chem. 265,17285-17293 and Ferry, et al. (1991) Proc. Natl. Acad. Sci. USA 88,8377-8381. The vector may be administered by injection, e.g.intravascularly or intramuscularly, inhalation, or other parenteralmode. Non-viral delivery methods such as administration of the DNA viacomplexes with liposomes or by injection, catheter or biolistics mayalso be used.

In yet another embodiment, cells are obtained from a subject, modifiedex vivo, and introduced into the same or a different subject. Additionalmethods of administration of the therapeutic compounds are set forthbelow.

A. Toxicity:

Toxicity and therapeutic efficacy of compounds of the invention can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., for determining The Ld50 (The Dose Lethal To50% Of The Population) And The Ed₅₀ (the dose therapeutically effectivein 50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index and it can be expressed as the ratioLD₅₀/ED₅₀. Compounds which exhibit large therapeutic indices arepreferred. While compounds that exhibit toxic side effects may be used,care should be taken to design a delivery system that targets suchcompounds to the site of affected tissue in order to minimize potentialdamage to uninfected cells and, thereby, reduce side effects.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. In particular,where the therapeutic is administered for potentiating AChR aggregation,it is desirable to establish the dose that will result in stimulation,if desired, or inhibition, if desired. Tests can then be continued inmedical tests. The dosage of such compounds lies preferably within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the method of the invention, the therapeuticallyeffective dose can be estimated initially from cell culture assays. Adose may be formulated in animal models to achieve a circulating plasmaconcentration range that includes the IC₅₀ (i.e., the concentration ofthe test compound which achieves a half-maximal inhibition of symptoms)as determined in cell culture. Such information can be used to moreaccurately determine useful doses in humans. Levels in plasma may bemeasured, for example, by high performance liquid chromatography.

B. Pharmaceutical Compositions:

Pharmaceutical compositions for use in accordance with the presentinvention may be formulated in conventional manner using one or morephysiologically acceptable carriers or excipients. Thus, the compoundsand their physiologically acceptable salts and solvates may beformulated for administration by, for example, injection, inhalation orinsufflation (either through the mouth or the nose) or oral, buccal,parenteral or rectal administration.

For such therapy, the compounds of the invention can be formulated for avariety of loads of administration, including systemic and topical orlocalized administration. Techniques and formulations generally may befound in Remmington's Pharmaceutical Sciences, Meade Publishing Co.,Easton, Pa. For systemic administration, injection is preferred,including intramuscular, intravenous, intraperitoneal, and subcutaneous.For injection, the compounds of the invention can be formulated inliquid solutions, preferably in physiologically compatible buffers suchas Hank's solution or Ringer's solution. In addition, the compounds maybe formulated in solid form and redissolved or suspended immediatelyprior to use. Lyophilized forms are also included.

For oral administration, the pharmaceutical compositions may take theform of, for example, tablets or capsules prepared by conventional meanswith pharmaceutically acceptable excipients such as binding agents(e.g., pregelatinised maize starch, polyvinylpyrrolidone orhydroxypropyl methylcellulose); fillers (e.g., lactose, microcrystallinecellulose or calcium hydrogen phosphate); lubricants (e.g., magnesiumstearate, talc or silica); disintegrants (e.g., potato starch or sodiumstarch glycolate); or wetting agents (e.g., sodium lauryl sulphate). Thetablets may be coated by methods well known in the art. Liquidpreparations for oral administration may take the form of, for example,solutions, syrups or suspensions, or they may be presented as a dryproduct for constitution with water or other suitable vehicle beforeuse. Such liquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., ationd oil, oily esters, ethyl alcohol or fractionated vegetableoils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates orsorbic acid). The preparations may also contain buffer salts, flavoring,coloring and sweetening agents as appropriate.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound. For buccal administration thecompositions may take the form of tablets or lozenges formulated inconventional manner. For administration by inhalation, the compounds foruse according to the present invention are conveniently delivered in theform of an aerosol spray presentation from pressurized packs or anebuliser, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof e.g., gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the compound and a suitable powder base suchas lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration bile salts and fusidic acidderivatives. In addition, detergents may be used to facilitatepermeation. Transmucosal administration may be through nasal sprays orusing suppositories. For topical administration, the oligomers of theinvention are formulated into ointments, salves, gels, or creams asgenerally known in the art. A wash solution can be used locally to treatan injury or inflammation to accelerate healing.

In clinical settings, a gene delivery system for the therapeutic geneencoding a proteoglycan of the invention can be introduced into apatient by any of a number of methods, each of which is familiar in theart. For instance, a pharmaceutical preparation of the gene deliverysystem can be introduced systemically, e.g., by intravenous injection,and specific transduction of the protein in the target cells occurspredominantly from specificity of transfection provided by the genedelivery vehicle, cell-type or tissue-type expression due to thetranscriptional regulatory sequences controlling expression of thereceptor gene, or a combination thereof. In other embodiments, initialdelivery of the recombinant gene is more limited with introduction intothe animal being quite localized. For example, the gene delivery vehiclecan be introduced by catheter (see U.S. Pat. No. 5,328,470) or bystereotactic injection (e.g., Chen et al. (1994) PNAS 91: 3054-3057). Agene encoding a proteoglycan of the invention can be delivered in a genetherapy construct by electroporation using techniques described, forexample, by Dev et al. ((1994) Cancer Treat Rev 20:105-115).

A preferred mode of delivering DNA to muscle cells include usingrecombinant adeno-associated virus vectors, such as those described inU.S. Pat. No. 5,858,351. Alternatively, genes have been delivered tomuscle by direct injection of plasmid DNA, such as described by Wolff etal. (1990) Science 247:1465-1468; Acsadi et al. (1991) Nature352:815-818; Barr and Leiden (1991) Science 254:1507-1509. However, thismode of administration generally results in sustained but generally lowlevels of expression. Low but sustained expression levels are expectedto be effective for practicing the methods of the invention.

The pharmaceutical preparation of the gene therapy construct or compoundof the invention can consist essentially of the gene delivery system inan acceptable diluent, or can comprise a slow release matrix in whichthe gene delivery vehicle or compound is imbedded. Alternatively, wherethe complete gene delivery system can be produced intact fromrecombinant cells, e.g., retroviral vectors, the pharmaceuticalpreparation can comprise one or more cells which produce the genedelivery system.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

VI. Diagnostic Methods

Based at least on the observation that biglycan binds to at least onecomponent of DAPCs, protein complexes which are critical for maintainingthe integrity of plasma membranes, the invention provides diagnosticmethods for determining whether a subject has or is likely to develop adisease or condition which is characterized by, or associated with,plasma membrane instability, in particular, abnormal or unstable DAPCs,such as muscular dystrophies. Furthermore, it has been observed in ananimal model for muscular dystrophy, which lacks dystrophin, that theamount of the proteoglycan biglycan is elevated, and thereby believed tobe a compensatory mechanism.

Furthermore, based at least on the observation that biglycan binds to,and phosphorylates MuSK and potentiates agrin-induced MuSKphosphorylation, and that biglycan stimulates agrin-mediated AChRaggregation, the invention also provides diagnostic methods fordetermining whether a subject has or is likely to develop a disease orcondition which is characterized by abnormal synapses or neuromuscularjunctions, e.g., neurological or neuromuscular diseases.

Accordingly, the identification of abnormal levels or activity of theproteoglycan of the invention in a subject would indicate that thesubject has, or is likely to develop a disease or condition relating toabnormal or unstable DAPCs. Diseases can be characterized by a highlevels of proteoglycan of the invention, e.g., if the cell compensatesfor the lack of another DAPC component or molecule associatingtherewith, e.g., as seen in dystrophin negative mice. Alternatively, ahigh level or activity of proteoglycan of the invention can at least bepart of the cause of the disease.

In addition, an elevated level or activity of a proteoglycan of theinvention could be associated with, or be at least in part, the cause ofneurological or neuromuscular diseases, e.g., by overstimulating AChRaggregation and/or activating MuSK.

Diseases are also likely to be caused or associated with a lower levelor activity of proteoglycan of the invention, which may, e.g., causeDAPCs to be more unstable than those on cells of subjects having anormal amount or activity of the proteoglycan of the invention.Accordingly, a lower level or activity of the proteoglycan of theinvention in cells of a subject would result in leaky membranes.

A lower level or activity of the proteoglycan of the invention couldalso result in insufficient AChR aggregation and/or insufficient MuSKactivation, thereby resulting in abnormal synapses or neuromuscularjunctions. Such situations can thus result in neurological orneuromuscular diseases, and result, e.g., in atrophy of tissues.

As used herein, the term “diagnostic assay” refers to the specific useof the methods described herein to identify an individual predisposed toa disease, such as a muscular disorder, a neuromuscular disorder or aneurological disorder. Such diagnostic assays are particularly useful asprenatal diagnostic assays, which can be used to determine whether afetus is predisposed to one or more of these disorders. For prenataldiagnosis, for example, a sample can be obtained by biopsy of muscletissue from the fetus or by biopsy of placenta from the pregnant mother.

In one embodiment, the method comprises determining the level of, or thebiological activity of a proteoglycan of the invention relative to thatin non affected subjects, or determining whether the proteoglycan orgene encoding it contains a mutation, or abnormal glycan side chains.

A patient sample may be any cell, tissue, or body fluid but ispreferably muscle tissue, cerebrospinal fluid, blood, or a bloodfraction such as serum or plasma. As used herein, the term “sample”refers to a specimen obtained from a subject, which can be a humansubject. In general, a tissue sample, which can be obtained, forexample, by biopsy of muscle or placenta of an individual suspected ofbeing predisposed to a disorder, is a suitable sample. In many cases, itis useful to prepare the sample as a tissue section, which can beexamined by histologic analysis. Alternatively, proteins or nucleicacids can be extracted from a sample and can be examined using methodssuch as gel electrophoresis and appropriate “blotting” methods, whichare well known in the art and described in detail below.

A sample can be obtained from a normal subject or from a test subject,who is suspected of being predisposed to a disorder, such as a muscular,neuromuscular or neurological disorder, and is being examined foraltered expression or localization of the proteoglycan of the inventionor altered expression of the mRNA encoding the proteoglycan of theinvention.

A sample obtained from a normal subject can be used as a “control”sample, which is useful for comparison with a sample obtained from atest subject. A control sample can be, for example, a muscle sample or aplacenta sample, which is obtained from an age- and sex-matchedindividual who does not exhibit and is not predisposed to a disorder,such as a muscular, neuromuscular, or neurological disorder. A controlsample exhibits a level of expression and a pattern of expression of theproteoglycan of the invention and a level of expression of theproteoglycan mRNA that is characteristic of the human population ingeneral and does not significantly deviate from the normal levels ofexpression or pattern of localization expected for a person in thepopulation. It is expected that, after a statistically significantnumber of control samples have been examined, an amount of expression ofthe proteoglycan of the invention per unit of a sample will bedetermined to be normal for a control sample. As used herein, a “normal”amount of proteoglycan of the invention in a control sample means anamount that is within an expected range for a person that is notpredisposed to a disorder, e.g., a muscular, neuromuscular, orneurological disorder.

Altered expression of the proteoglycan of the invention in a sampleobtained from a test subject can be identified qualitatively by visuallycomparing, for example, photomicrographs of an immunohistochemicallystained control sample with the sample obtained from the test subject.Alternatively, altered expression of proteoglycan of the invention canbe measured quantitatively using, for example, densitometric analysis.Altered expression of proteoglycan of the invention protein also can bedetermined using methods of gel electrophoresis and, if desired,immunoblot analysis. Such methods are well known in the art.

In the diagnostic method of the present invention, a muscle biopsysample is obtained from an individual to be tested. Typically, anindividual to be tested according to the diagnostic assays of theinvention is an individual who is at risk of having a disorder, e.g., amuscular, neuromuscular, or neurological disorder, for example, a personfrom a family at risk, or a person showing one or more symptoms of suchdisorders. In the case of muscular or neuromuscular disorders, musclesamples can be obtained from patients by surgical biopsy. The site ofbiopsy can be any skeletal muscle suspected of being dystrophic. Musclegroups about the shoulder and pelvic girdles, however, are the mostaffected and are likely to be the most common site of biopsy. Suchmuscle samples are analyzed for the presence and/or biological activityof the proteoglycan of the invention, and can also be analyzed byantibody staining to determine levels of dystrophin,dystrophin-associated proteins. To ensure that control and experimentalextracts contain substantially similar quantities of protein, extractsare separated electrophoretically and stained, for example, withCoomassie blue.

Methods for the determination of levels of dystrophin anddystrophin-associated proteins are carried out by conventionaltechniques. Such techniques are disclosed, for example, in U.S. Pat.Nos. 5,187,063; 5,260,209; and 5,308,752, the disclosures of which areincorporated herein by reference. International Publication Number WO89/06286 also discloses such conventional techniques, as well as thenucleic sequence encoding dystrophin.

Altered localization of the proteoglycan of the invention in a samplealso can be determined. As used herein, the term “localization” refersto the pattern of deposition of the proteoglycan of the invention in asample. The localization of the proteoglycan of the invention also canbe determined qualitatively or quantitatively. “Altered” localizationrefers to a pattern of deposition of the proteoglycan of the inventionin a sample that is different from the pattern of localization observedin a control sample.

The level of expression mRNA encoding the proteoglycan of the inventioncan be determined and can be used to identify an individual that ispredisposed to a disorder, such as muscular, neuromuscular, orneurological disorder. Methods for determining the level of expressionof proteoglycan mRNA in a sample are well known in the art and include,for example, northern blot analysis, which can be used to determinewhether proteoglycan mRNA is expressed at a normal level in a testsample. Northern blot analysis also can be used to determine whether theproteoglycan mRNA that is expressed in a cell is a full lengthtranscript. For example, an RNA sample obtained from a tissue sample canbe contacted with a nucleic acid probe that hybridizes to the mRNAencoding the proteoglycan of the invention. One skilled in the art wouldknow that the probe can be a DNA or RNA probe and can be prepared from acDNA encoding the proteoglycan or can be synthesized as anoligonucleotide. In addition, the skilled artisan would recognize thatsuch hybridization should be performed under stringent conditions, whichcan be determined empirically (see, for example, Sambrook et al.,Molecular Cloning: A laboratory manual (Cold Spring Harbor LaboratoryPress 1989), which is incorporated herein by reference). Methods forisolating intact total RNA and poly A+ mRNA and for performing Northernblot analysis are well known in the art (Sambrook et al., 1989).

A sensitive method of determining the level of expression of mRNAencoding the proteoglycan of the invention in a sample is the reversetranscriptase-polymerase chain reaction (RT-PCR), which is well known inthe art (see, for example, H. A. Erlich, PCR Technology: Principles andapplications for DNA amplification (Stockton Press, 1989), which isincorporated herein by reference; see chap. 8). The RT-PCR method isparticularly useful for examining a sample that fails to give adetectable signal by northern blot analysis. Due to the amplificationsteps involved in PCR analysis, a rare proteoglycan mRNA can beidentified in a sample.

Methods for determining levels of proteoglycan of the invention can use,e.g., antibodies binding to the proteoglycan of the invention. Anantibody can be used in connection with a conventional assay for thedetermination of levels of antigen in a tissue of interest, e.g., muscletissue. Any method which enables the determination of protein levelspresent in muscle tissue based on antibody binding is useful inconnection with the present invention. Preferred methods include Westernblotting, immunocytochemical analysis and enzyme-linked immunoadsorbentassay (ELISA).

For assays which require solubilized extracellular matrix (e.g., ELISAand Western blotting), the amount of muscle obtained by biopsy should besufficient to enable the extraction of the proteoglycan of the inventionin a quantity sufficient for analysis. In an illustrative embodiment,the muscle tissue is homogenized by mechanical disruption using anapparatus such as a hand operated or motor driven glass homogenizer, aWaring blade blender homogenizer, or an ultrasonic probe. Homogenizationcan be carried out, for example, in a buffer having a pH of about 11 or12, as further described in the Examples. The buffer can furthercomprise protease inhibitors, e.g., 1 mM PMSF, 0.75 mM benzamidine, 1 mug/ml aprotinin, 1 mu g/ml of leupeptin, 1 mu g/ml of pepstatin A. Theincubation is then carried out, e.g., on ice for 2 hr. Followingcentrifugation, extracellular matrix solubilized in this manner can thenbe processed by conventional methods for use, for example, in Westernblotting or ELISA analytical formats.

The solubilized extracellular matrix components, prepared as describedabove can be analyzed by Western blotting by first separating thecomponents on a 3-12% SDS polyacrylamide gel (Laemmli (1970) Nature 227,680) followed by transfer to a solid support, such as a nitrocellulosemembrane, forming an exact replica of the original protein separationbut leaving the transferred proteins accessible for further study. Thissolid support bearing the transferred protein components is referred toas an immunoblot. The detection of transferred proteins can beaccomplished by the use of general protein dyes such as Amido black orCoomassie brilliant blue. Antibodies which are specific for theproteoglycan of the invention can be labeled with a detectable reportergroup and used to stain the protein transferred to the solid support.Alternatively, unlabeled antibodies specific for the proteoglycan of theinvention are incubated with an immunoblot under conditions appropriatefor binding. The specific binding of these antibodies to the muscletissue sample can be detected through the use of labeled secondaryantibodies by conventional techniques.

The methods of the present invention can also be practiced in anenzyme-linked immunoadsorbent assay (ELISA) format. In this format,antibodies against the proteoglycan of the invention are adsorbed to asolid support, in most cases a polystyrene microtiter plate. Aftercoating the support with antibody and washing, a solubilized sample isadded. Proteoglycan of the invention, if present, will bind to theadsorbed antibodies. Next, a conjugate that will also bind to theproteoglycan of interest is added. A conjugates can be an antibodymolecule which binds to the proteoglycan of the invention, and to whichan enzyme is covalently bound. After addition of a chromogenic substratefor the enzyme, the intensity of the colored reaction products generatedwill be proportional to the amount of conjugated enzyme and thusindirectly to the amount of bound proteoglycan of the invention. Sincethe intensity of the developed color is proportional to the amount ofproteoglycan of the invention present, determination of the intensity ofthe color produced by a standard series of concentrations ofproteoglycan of the invention will allow the calculation of the amountof proteoglycan of the invention in an unknown sample. Many variationsof this assay exist as described in Voller, A., Bidwell, D. E. andBartlett, A., The Enzyme Linked Immunoadsorbent Assay (ELISA): A guidewith abstracts of microplate applications, Dynatech Laboratories,Alexandria, Va. (1979).

Alternatively, tissue specimens (e.g., human biopsy samples) can betested for the presence of the components of the DAPC complex by usingmonoclonal or polyclonal antibodies in an immunohistochemical technique,such as the immunoperoxidase staining procedure. In addition,immunofluorescent techniques can be used to examine human tissuespecimens. In a typical protocol, slides containing cryostat sections offrozen, unfixed tissue biopsy samples are air-dried and then incubatedwith an antibody preparation against the proteoglycan (primary antibody)of the invention in a humidified chamber at room temperature. The slidesare layered with a preparation of fluorescently labeled antibodydirected against the primary antibody. Labeled secondary antibodies arealso useful for detection. The staining pattern and intensities withinthe sample can be determined by fluorescent light microscopy.

The invention also provides a prenatal diagnostic screening procedureusing a tissue such as placenta or fetal muscle, wherein the screeningprocedure is useful for identifying an individual predisposed to adisorder, such as a muscular, neuromuscular, or neurological disorder.

In preferred embodiments, the methods for determining whether a subjecthas or is at risk for developing a disease, such as a muscular,neuromuscular, or neurological disease, is characterized as comprisingdetecting, in a tissue sample or in cells of the subject, the presenceor absence of a genetic alteration characterized by at least one of (i)an alteration affecting the integrity of a gene encoding a proteoglycanof the invention, or (ii) the mis-expression of a gene encoding aproteoglycan of the invention. To illustrate, such genetic alterationscan be detected by ascertaining the existence of at least one of (i) adeletion of one or more nucleotides from a gene encoding a proteoglycanof the invention, (ii) an addition of one or more nucleotides to a geneencoding a proteoglycan of the invention, (iii) a substitution of one ormore nucleotides of a gene encoding a proteoglycan of the invention,(iv) a gross chromosomal rearrangement of a gene encoding a proteoglycanof the invention, (v) a gross alteration in the level of a messenger RNAtranscript of a gene encoding a proteoglycan of the invention, (vii)aberrant modification of a gene encoding a proteoglycan of theinvention, such as of the methylation pattern of the genomic DNA, (vii)the presence of a non-wild type splicing pattern of a messenger RNAtranscript of a gene encoding a proteoglycan of the invention, (viii) anon-wild type level of proteoglycan of the invention, (ix) allelic lossof a gene encoding a proteoglycan of the invention, and/or (x)inappropriate post-translational modification of a proteoglycan of theinvention, such as the presence of abnormal glycosamino glycan sidechains. As set out below, the present invention provides a large numberof assay techniques for detecting alterations in a gene encoding aproteoglycan of the invention. These methods include, but are notlimited to, methods involving sequence analysis, Southern blothybridization, restriction enzyme site mapping, and methods involvingdetection of absence of nucleotide pairing between the nucleic acid tobe analyzed and a probe. These and other methods are further describedinfra.

Specific diseases or disorders, e.g., genetic diseases or disorders, areassociated with specific allelic variants of polymorphic regions ofcertain genes, which do not necessarily encode a mutated protein. Thus,the presence of a specific allelic variant of a polymorphic region of agene, such as a single nucleotide polymorphism (“SNP”), in a subject canrender the subject susceptible to developing a specific disease ordisorder. Polymorphic regions in genes, e.g., a gene encoding aproteoglycan of the invention, can be identified, by determining thenucleotide sequence of genes in populations of individuals. If apolymorphic region, e.g., SNP, is identified, then the link with aspecific disease can be determined by studying specific populations ofindividuals, e.g, individuals which developed a specific disease, such amuscular, neuromuscular, or neurological disease. A polymorphic regioncan be located in any region of a gene, e.g., exons, in coding or noncoding regions of exons, introns, and promoter region.

It is likely that genes encoding proteoglycans of the invention comprisepolymorphic regions, specific alleles of which may be associated withspecific diseases or conditions or with an increased likelihood ofdeveloping such diseases or conditions. Thus, the invention providesmethods for determining the identity of the allele or allelic variant ofa polymorphic region of a gene encoding a proteoglycan of the inventionin a subject, to thereby determine whether the subject has or is at riskof developing a disease or disorder associated with a specific allelicvariant of a polymorphic region.

In an exemplary embodiment, there is provided a nucleic acid compositioncomprising a nucleic acid probe including a region of nucleotidesequence which is capable of hybridizing to a sense or antisensesequence of a gene encoding a proteoglycan of the invention or naturallyoccurring mutants thereof, or 5′ or 3′ flanking sequences or intronicsequences naturally associated with the subject proteoglycan genes ornaturally occurring mutants thereof. The nucleic acid of a cell isrendered accessible for hybridization, the probe is contacted with thenucleic acid of the sample, and the hybridization of the probe to thesample nucleic acid is detected. Such techniques can be used to detectalterations or allelic variants at either the genomic or mRNA level,including deletions, substitutions, etc., as well as to determine mRNAtranscript levels.

A preferred detection method is allele specific hybridization usingprobes overlapping the mutation or polymorphic site and having about 5,10, 20, 25, or 30 nucleotides around the mutation or polymorphic region.In a preferred embodiment of the invention, several probes capable ofhybridizing specifically to allelic variants, such as single nucleotidepolymorphisms, are attached to a solid phase support, e.g., a “chip”.Oligonucleotides can be bound to a solid support by a variety ofprocesses, including lithography. For example a chip can hold up to250,000 oligonucleotides. Mutation detection analysis using these chipscomprising oligonucleotides, also termed “DNA probe arrays” is describede.g., in Cronin et al. (1996) Human Mutation 7:244. In one embodiment, achip comprises all the allelic variants of at least one polymorphicregion of a gene. The solid phase support is then contacted with a testnucleic acid and hybridization to the specific probes is detected.Accordingly, the identity of numerous allelic variants of one or moregenes can be identified in a simple hybridization experiment.

In certain embodiments, detection of the alteration comprises utilizingthe probe/primer in a polymerase chain reaction (PCR) (see, e.g. U.S.Pat. Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or,alternatively, in a ligase chain reaction (LCR) (see, e.g., Landegran etal. (1988) Science 241:1077-1080; and Nakazawa et al. (1994) PNAS91:360-364), the latter of which can be particularly useful fordetecting point mutations in the gene (see Abravaya et al. (1995) NucAcid Res 23:675-682). In a merely illustrative embodiment, the methodincludes the steps of (i) collecting a tissue or cell sample from apatient, (ii) isolating nucleic acid (e.g., genomic, mRNA or both) fromthe cells of the sample, (iii) contacting the nucleic acid sample withone or more primers which specifically hybridize to the gene of interest(i.e., encoding the proteoglycan of interest) under conditions such thathybridization and amplification of the gene (if present) occurs, and(iv) detecting the presence or absence of an amplification product, ordetecting the size of the amplification product and comparing the lengthto a control sample. It is anticipated that PCR and/or LCR may bedesirable to use as a preliminary amplification step in conjunction withany of the techniques used for detecting mutations described herein.

Alternative amplification methods include: self sustained sequencereplication (Guatelli, J. C. et al., 1990, Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh, D. Y. et al.,1989, Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase(Lizardi, P. M. et al., 1988, Bio/Technology 6:1197), or any othernucleic acid amplification method, followed by the detection of theamplified molecules using techniques well known to those of skill in theart. These detection schemes are especially useful for the detection ofnucleic acid molecules if such molecules are present in very lownumbers.

In yet another embodiment, any of a variety of sequencing reactionsknown in the art can be used to directly sequence a gene of interest anddetect mutations by comparing the sequence of the sample gene with thecorresponding wild-type (control) sequence. Exemplary sequencingreactions include those based on techniques developed by Maxam andGilbert (Proc. Natl. Acad Sci USA (1977) 74:560) or Sanger (Sanger et al(1977) Proc. Nat. Acad. Sci. 74:5463). It is also contemplated that anyof a variety of automated sequencing procedures may be utilized whenperforming the subject assays (Biotechniques (1995) 19:448), includingsequencing by mass spectrometry (see, for example PCT publication WO94/16101; Cohen et al. (1996) Adv Chromatogr 36:127-162; and Griffin etal. (1993) Appl Biochem Biotechnol 38:147-159). It will be evident toone skilled in the art that, for certain embodiments, the occurrence ofonly one, two or three of the nucleic acid bases need be determined inthe sequencing reaction. For instance, A-track or the like, e.g., whereonly one nucleic acid is detected, can be carried out.

In a further embodiment, protection from cleavage agents (such as anuclease, hydroxylamine or osmium tetroxide and with piperidine) can beused to detect mismatched bases in RNA/RNA or RNA/DNA or DNA/DNAheteroduplexes (Myers, et al. (1985) Science 230:1242). In general, theart technique of “mismatch cleavage” starts by providing heteroduplexesformed by hybridizing (labelled) RNA or DNA containing the wild-typesequence with potentially mutant RNA or DNA obtained from a tissuesample. The double-stranded duplexes are treated with an agent whichcleaves single-stranded regions of the duplex such as which will existdue to base pair mismatches between the control and sample strands. Forinstance, RNA/DNA duplexes can be treated with RNase and DNA/DNA hybridstreated with S1 nuclease to enzymatically digest the mismatched regions.In other embodiments, either DNA/DNA or RNA/DNA duplexes can be treatedwith hydroxylamine or osmium tetroxide and with piperidine in order todigest mismatched regions. After digestion of the mismatched regions,the resulting material is then separated by size on denaturingpolyacrylamide gels to determine the site of mutation. See, for example,Cotton et al (1988) Proc. Nall Acad Sci USA 85:4397; Saleeba et al(1992) Methods Enzymod. 217:286-295. In a preferred embodiment, thecontrol DNA or RNA can be labeled for detection.

In still another embodiment, the mismatch cleavage reaction employs oneor more proteins that recognize mismatched base pairs in double-strandedDNA (so called “DNA mismatch repair” enzymes) in defined systems fordetecting and mapping point mutations in cDNAs obtained from samples ofcells. For example, the mutY enzyme of E. coli cleaves A at G/Amismatches and the thymidine DNA glycosylase from HeLa cells cleaves Tat G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).According to an exemplary embodiment, a probe based on a sequenceencoding a proteoglycan of the invention, e.g., a wild-type sequence, ishybridized to a cDNA or other DNA product from a test cell(s). Theduplex is treated with a DNA mismatch repair enzyme, and the cleavageproducts, if any, can be detected from electrophoresis protocols or thelike. See, for example, U.S. Pat. No. 5,459,039.

In other embodiments, alterations in electrophoretic mobility will beused to identify mutations or the identity of the allelic variant of apolymorphic region in genes. For example, single strand conformationpolymorphism (SSCP) may be used to detect differences in electrophoreticmobility between mutant and wild type nucleic acids (Orita et al. (1989)Proc Natl. Acad. Sci. USA 86:2766, see also Cotton (1993) Mutat Res285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).Single-stranded DNA fragments of sample and control nucleic acids willbe denatured and allowed to renature. The secondary structure ofsingle-stranded nucleic acids varies according to sequence, theresulting alteration in electrophoretic mobility enables the detectionof even a single base change. The DNA fragments may be labeled ordetected with labeled probes. The sensitivity of the assay may beenhanced by using RNA (rather than DNA), in which the secondarystructure is more sensitive to a change in sequence. In a preferredembodiment, the subject method utilizes heteroduplex analysis toseparate double stranded heteroduplex molecules on the basis of changesin electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).

Examples of other techniques for detecting point mutations or theidentity of the allelic variant of a polymorphic region include, but arenot limited to, selective oligonucleotide hybridization, selectiveamplification, or selective primer extension. For example,oligonucleotide primers may be prepared in which the known mutation ornucleotide difference (e.g., in allelic variants) is placed centrallyand then hybridized to target DNA under conditions which permithybridization only if a perfect match is found (Saiki et al. (1986)Nature 324:163); Saiki et al (1989) Proc. Natl. Acad. Sci. USA 86:6230).Such allele specific oligonucleotide hybridization techniques may beused to test one mutation or polymorphic region per reaction whenoligonucleotides are hybridized to PCR amplified target DNA or a numberof different mutations or polymorphic regions when the oligonucleotidesare attached to the hybridizing membrane and hybridized with labelledtarget DNA.

Yet other techniques that can be used to detect a mutation of specificallele include the following: selective PCR amplification as describedin Gibbs et al (1989) Nucleic Acids Res. 17:2437-2448, Prossner (1993)Tibtech 11:238, and Gasparini et al (1992) Mol. Cell Probes 6:1;oligonucleotide ligation assay (OLA), as described, e.g., in U.S. Pat.No. 4,998,617 and in Landegren, U. et al., Science 241:1077-1080 (1988),U.S. Pat. No. 5,593,826, Tobe et al. (1996) Nucleic Acids Res 24: 3728.Other techniques can be used for detecting a single nucleotidepolymorphism. Examples of such techniques are disclosed, e.g., in Mundy,C. R. U.S. Pat. No. 4,656,127; Cohen, D. et al. (French Patent2,650,840; PCT Appln. No. WO91/02087); Genetic Bit Analysis or GBA™,described by Goelet, P. et al. (PCT Appln. No. 92/15712). Komher, J. S.et al., Nucl. Acids. Res. 17:7779-7784 (1989); Sokolov, B. P., Nucl.Acids Res. 18:3671 (1990); Syvanen, A.-C., et al., Genomics 8:684-692(1990); Kuppuswamy, M. N. et al., Proc. Natl. Acad. Sci. (U.S.A.)88:1143-1147 (1991); Prezant, T. R. et al., Hum. Mutat. 1:159-164(1992); Ugozzoli, L. et al., GATA 9:107-112 (1992); Nyren, P. et al.,Anal. Biochem. 208:171-175 (1993)).

For mutations that produce premature termination of protein translation,the protein truncation test (PTT) offers an efficient diagnosticapproach (Roest, et. al., (1993) Hum. Mol. Genet. 2:1719-21; van derLuijt, et. al., (1994) Genomics 20:1-4).

The methods described herein may be performed, for example, by utilizingpre-packaged diagnostic kits comprising at least one probe nucleic acid,primer set; and/or antibody reagent described herein, which may beconveniently used, e.g., in clinical settings to diagnose patientsexhibiting symptoms or family history of a disease or illness involvinga proteoglycan of the invention (see below).

Any cell type or tissue may be utilized in the diagnostics describedbelow. In a preferred embodiment a bodily fluid, e.g., blood, isobtained from the subject to determine the presence of a mutation or theidentity of the allelic variant of a polymorphic region of a geneencoding a proteoglycan of interest. A bodily fluid, e.g, blood, can beobtained by known techniques (e.g. venipuncture). Alternatively, nucleicacid tests can be performed on dry samples (e.g. hair or skin). Forprenatal diagnosis, fetal nucleic acid samples can be obtained frommaternal blood as described in International Patent Application No.WO91/07660 to Bianchi. Alternatively, amniocytes or chorionic villi maybe obtained for performing prenatal testing.

Diagnostic procedures may also be performed in situ directly upon tissuesections (fixed and/or frozen) of patient tissue obtained from biopsiesor resections, such that no nucleic acid purification is necessary.Nucleic acid reagents may be used as probes and/or primers for such insitu procedures (see, for example, Nuovo, G.J., 1992, PCR in situHybridization: Protocols and Applications, Raven Press, NY).

In addition to methods which focus primarily on the detection of onenucleic acid sequence, profiles may also be assessed in such detectionschemes. Fingerprint profiles may be generated, for example, byutilizing a differential display procedure, Northern analysis and/orRT-PCR.

Antibodies directed against wild type or mutatnt proteoglycans of theinvention or allelic variant thereof, which are discussed above, mayalso be used in disease diagnostics and prognostics. Such diagnosticmethods, may be used to detect abnormalities in the level of theexpression of the proteoglycan of the invention, or abnormalities in thestructure and/or tissue, cellular, or subcellular location of theproteoglycan. Structural differences may include, for example,differences in the size, electronegativity, or antigenicity of themutant proteoglycan of the invention relative to the normalproteoglycan. Protein from the tissue or cell type to be analyzed mayeasily be detected or isolated using techniques which are well known toone of skill in the art, including but not limited to Western blotanalysis. For a detailed explanation of methods for carrying out Westernblot analysis, see Sambrook et al, 1989, supra, at Chapter 18. Theprotein detection and isolation methods employed herein may also be suchas those described in Harlow and Lane, for example, (Harlow, E. andLane, D., 1988, “Antibodies: A Laboratory Manual”, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.), which is incorporatedherein by reference in its entirety.

This can be accomplished, for example, by immunofluorescence techniquesemploying a fluorescently labeled antibody (see below) coupled withlight microscopic, flow cytometric, or fluorimetric detection. Theantibodies (or fragments thereof) useful in the present invention may,additionally, be employed histologically, as in immunofluorescence orimmunoelectron microscopy, for in situ detection of proteoglycans. Insitu detection may be accomplished by removing a histological specimenfrom a patient, and applying thereto a labeled antibody of the presentinvention. The antibody (or fragment) is preferably applied byoverlaying the labeled antibody (or fragment) onto a biological sample.Through the use of such a procedure, it is possible to determine notonly the presence of a proteoglycan of the invention, but also itsdistribution in the examined tissue. Using the present invention, one ofordinary skill will readily perceive that any of a wide variety ofhistological methods (such as staining procedures) can be modified inorder to achieve such in situ detection.

Often a solid phase support or carrier is used as a support capable ofbinding an antigen or an antibody. Well-known supports or carriersinclude glass, polystyrene, polypropylene, polyethylene, dextran, nylon,amylases, natural and modified celluloses, polyacrylamides, gabbros, andmagnetite. The nature of the carrier can be either soluble to someextent or insoluble for the purposes of the present invention. Thesupport material may have virtually any possible structuralconfiguration so long as the coupled molecule is capable of binding toan antigen or antibody. Thus, the support configuration may bespherical, as in a bead, or cylindrical, as in the inside surface of atest tube, or the external surface of a rod. Alternatively, the surfacemay be flat such as a sheet, test strip, etc. Preferred supports includepolystyrene beads. Those skilled in the art will know many othersuitable carriers for binding antibody or antigen, or will be able toascertain the same by use of routine experimentation.

One means for labeling an antibody that specifically binds to aproteoglycan of the invention is via linkage to an enzyme and use in anenzyme immunoassay (EIA) (Voller, “The Enzyme Linked Immunosorbent Assay(ELISA)”, Diagnostic Horizons 2:1-7, 1978, Microbiological AssociatesQuarterly Publication, Walkersville, Md.; Voller, et al., J. Clin.Pathol. 31:507-520 (1978); Butler, Meth. Enzymol. 73:482-523 (1981);Maggio, (ed.) Enzyme Immunoassay, CRC Press, Boca Raton, Fla., 1980;Ishikawa, et al., (eds.) Enzyme Immunoassay, Kgaku Shoin, Tokyo, 1981).The enzyme which is bound to the antibody will react with an appropriatesubstrate, preferably a chromogenic substrate, in such a manner as toproduce a chemical moiety which can be detected, for example, byspectrophotometric, fluorimetric or by visual means. Enzymes which canbe used to detectably label the antibody include, but are not limitedto, malate dehydrogenase, staphylococcal nuclease, delta-5-steroidisomerase, yeast alcohol dehydrogenase, α-glycerophosphate,dehydrogenase, triose phosphate isomerase, horseradish peroxidase,alkaline phosphatase, asparaginase, glucose oxidase, beta-galactosidase,ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase,glucoamylase and acetylcholinesterase. The detection can be accomplishedby colorimetric methods which employ a chromogenic substrate for theenzyme. Detection may also be accomplished by visual comparison of theextent of enzymatic reaction of a substrate in comparison with similarlyprepared standards.

Detection may also be accomplished using any of a variety of otherimmunoassays. For example, by radioactively labeling the antibodies orantibody fragments, it is possible to detect fingerprint gene wild typeor mutant peptides through the use of a radioimmunoassay (RIA) (see, forexample, Weintraub, B., Principles of Radioimmunoassays, SeventhTraining Course on Radioligand Assay Techniques, The Endocrine Society,March, 1986, which is incorporated by reference herein). The radioactiveisotope can be detected by such means as the use of a gamma counter or ascintillation counter or by autoradiography.

It is also possible to label the antibody with a fluorescent compound.When the fluorescently labeled antibody is exposed to light of theproper wave length, its presence can then be detected due tofluorescence. Among the most commonly used fluorescent labelingcompounds are fluorescein isothiocyanate, rhodamine, phycoerythrin,phycocyanin, allophycocyanin, o-phthaldehyde and fluorescamine.

The antibody can also be detectably labeled using fluorescence emittingmetals such as ¹⁵²Eu, or others of the lanthanide series. These metalscan be attached to the antibody using such metal chelating groups asdiethylenetriaminepentacetic acid (DTPA) or ethylenediaminetetraaceticacid (EDTA).

The antibody also can be detectably labeled by coupling it to achemiluminescent compound. The presence of the chemiluminescent-taggedantibody is then determined by detecting the presence of luminescencethat arises during the course of a chemical reaction. Examples ofparticularly useful chemiluminescent labeling compounds are luminol,isoluminol, theromatic acridinium ester, imidazole, acridinium salt andoxalate ester.

Likewise, a bioluminescent compound may be used to label the antibody ofthe present invention. Bioluminescence is a type of chemiluminescencefound in biological systems in, which a catalytic protein increases theefficiency of the chemiluminescent reaction. The presence of abioluminescent protein is determined by detecting the presence ofluminescence. Important bioluminescent compounds for purposes oflabeling are luciferin, luciferase and aequorin.

Moreover, it will be understood that any of the above methods fordetecting alterations in a gene or gene product or polymorphic variantscan be used to monitor the course of treatment or therapy.

VII. Screening Methods

The invention further provides methods for identifying agents, e.g.,bigylcan therapeutics, which modulate membrane integrity, in particular,by modulating DAPC stability, and agents which modulate neuromuscularjunction formation, such as by modulating postsynaptic differentiation.Thus, the invention provides methods for identifying agents whichmodulate the activity of a proteoglycan of the invention, e.g., DAG-125,or proteoglycan having similar activity. The agent can be an agonist ofa biological activity of a proteoglycan of the invention, or the agentcan be an antagonist of a proteoglycan of the invention. An agonistagent will be of interest for use in prophylactic and therapeutictreatments of diseases or disorders, e.g., characterized by an instableDAPC or an inappropriate formation of a postsynaptic differentiation. Anantagonist agent will be of interest for use in prophylactic andtherapeutic treatments of diseases or disorders, e.g., characterized byan overactive neuromuscular junction, e.g., in situations in which thereis an excess of the proteoglycan of the invention.

Accordingly, the invention provides screening methods for identifyingtherapeutics. A therapeutic of the invention can be any type ofcompound, including a protein, a peptide, a proteoglycan, apolysaccharide, a peptidomimetic, a small molecule, and a nucleic acid.A nucleic acid can be, e.g., a gene, an antisense nucleic acid, aribozyme, or a triplex molecule.

Preferred agonists include compounds, such as proteoglycans, which mimicat least one biological activity of a proteoglycan of the invention,e.g., the capability to bind to one or more components of a DAPC, suchas alpha-dystroglycan, or the capability to stimulate MuSKphosphorylation and/or AChR aggregation. Other preferred agonistsinclude compounds which are capable of increasing the production of theproteoglycan of the invention in a cell, e.g., compounds capable ofupregulating the expression of the gene encoding the proteoglycan, andcompounds which are capable of enhancing an activity of a proteoglycanof the invention, and/or the interaction of a proteoglycan of theinvention with another molecule, such as a component of a DAPC or MuSK.

Preferred antagonists include compounds which are dominant negativeproteins, which, e.g., are capable of binding to α-sarcoglycan, but notto stabilize DAPCs, such as by competing with the endogenousproteoglycan of the invention. Other preferred antagonists includecompounds which decrease or inhibit the production of a proteoglycan ofthe invention in a cell and compounds which are capable ofdownregulating expression of a gene encoding a proteoglycan of theinvention, and compounds which are capable of donwregulating an activityof a proteoglycan of the invention and/or its interaction with anothermolecule, such as α-sarcoglycan. In another preferred embodiment, anantagonist is a modified form of an alpha-dystroglycan or other moleculecapable of binding to the wildtype proteoglycan of the invention, whichis capable of interacting with the proteoglycan of the invention, butwhich does not have biological activity, e.g., which does not stabilizeDAPCs.

The invention also provides screening methods for identifyingtherapeutics which are capable of binding to a proteoglycan of theinvention, e.g., a wild-type proteoglycan of the invention or a mutatedform thereof, and thereby modulate the a biological activity of aproteoglycan of the invention, or degrades, or causes the proteoglycanof the invention to be degraded. For example, such a therapeutic can bean antibody or derivative thereof which interacts specifically with aproteoglycan of the invention (either wild-type or mutated).

Thus, the invention provides screening methods for identifying agonistand antagonist compounds, comprising selecting compounds which arecapable of interacting with a proteoglycan of the invention or with amolecule interacting with a proteoglycan of the invention, such acomponent of a DAPC or MuSK, and/or compounds which are capable ofmodulating the interaction of an a proteoglycan of the invention withanother molecule, such as a component of a DAPC or MuSK. In general, amolecule which is capable of interacting with a proteoglycan of theinvention is referred to herein as “proteoglycan binding partner” or“PT-binding partner” and can be a component of a DAPC, e.g., adystroglycan or a sarcoglycan, or MuSK.

The compounds of the invention can be identified using various assaysdepending on the type of compound and activity of the compound that isdesired. Set forth below are at least some assays that can be used foridentifying therapeutics of the invention. It is within the skill of theart to design additional assays for identifying therapeutics.

A. Cell-Free Assays

Cell-free assays can be used to identify compounds which are capable ofinteracting with a proteoglycan of the invention or binding partnerthereof, to thereby modify the activity of the proteoglycan of theinvention or binding partner thereof. Such a compound can, e.g., modifythe structure of a proteoglycan of the invention or binding partnerthereof and thereby affect its activity. Cell-free assays can also beused to identify compounds which modulate the interaction between aproteoglycan of the invention and a PT-binding partner, such as acomponent of a DAPC. In a preferred embodiment, cell-free assays foridentifying such compounds consist essentially in a reaction mixturecontaining a proteoglycan of the invention, and a test compound or alibrary of test compounds with or without a binding partner. A testcompound can be, e.g., a derivative of a PT-binding partner, e.g., anbiologically inactive target peptide, or a small molecule.

These assays can be performed with a complete proteoglycan molecule ofthe invention. Alternatively, the screening assays can be performed withpotions thereof, such as the core only, one or more LLR domains, theglycosamino glycan chains only, or portions thereof, or combinations ofthese portions. These can be prepared as set forth supra.

Accordingly, one exemplary screening assay of the present inventionincludes the steps of contacting a proteoglycan of the invention orfunctional fragment thereof or a PT-binding partner with a test compoundor library of test compounds and detecting the formation of complexes.For detection purposes, the molecule can be labeled with a specificmarker and the test compound or library of test compounds labeled with adifferent marker. Interaction of a test compound with a proteoglycan ofthe invention or fragment thereof or PT-binding partner can then bedetected by determining the level of the two labels after an incubationstep and a washing step. The presence of two labels after the washingstep is indicative of an interaction.

An interaction between molecules can also be identified by usingreal-time BIA (Biomolecular Interaction Analysis, Pharmacia BiosensorAB) which detects surface plasmon resonance (SPR), an opticalphenomenon. Detection depends on changes in the mass concentration ofmacromolecules at the biospecific interface, and does not require anylabeling of interactants. In one embodiment, a library of test compoundscan be immobilized on a sensor surface, e.g., which forms one wall of amicro-flow cell. A solution containing the proteoglycan of theinvention, functional fragment thereof, analog or PT-binding partner isthen flown continuously over the sensor surface. A change in theresonance angle as shown on a signal recording, indicates that aninteraction has occurred. This technique is further described, e.g., inBIAtechnology Handbook by Pharmacia.

Another exemplary screening assay of the present invention includes thesteps of (a) forming a reaction mixture including: (i) a proteoglycan ofthe invention, (ii) a PT-binding partner (e.g., α-sarcoglycan), and(iii) a test compound; and (b) detecting interaction of the proteoglycanof the invention and the PT-binding protein. The proteoglycan of theinvention and PT-binding partner can be produced recombinantly, purifiedfrom a source, e.g., plasma, or chemically synthesized, as describedherein. A statistically significant change (potentiation or inhibition)in the interaction of the proteoglycan of the invention and PT-bindingprotein in the presence of the test compound, relative to theinteraction in the absence of the test compound, indicates a potentialagonist (mimetic or potentiator) or antagonist (inhibitor) of abioactivity for the test compound. The compounds of this assay can becontacted simultaneously. Alternatively, a proteoglycan of the inventioncan first be contacted with a test compound for an appropriate amount oftime, following which the PT-binding partner is added to the reactionmixture. The efficacy of the compound can be assessed by generating doseresponse curves from data obtained using various concentrations of thetest compound. Moreover, a control assay can also be performed toprovide a baseline for comparison. In the control assay, isolated andpurified proteoglycan of the invention or binding partner is added to acomposition containing the PT-binding partner or proteoglycan of theinvention, and the formation of a complex is quantitated in the absenceof the test compound.

Complex formation between a proteoglycan of the invention and aPT-binding partner may be detected by a variety of techniques.Modulation of the formation of complexes can be quantitated using, forexample, detectably labeled proteins such as radiolabeled, fluorescentlylabeled, or enzymatically labeled proteoglycans of the invention orPT-binding partners, by immunoassay, or by chromatographic detection.

Typically, it will be desirable to immobilize either the proteoglycan ofthe invention or its binding partner to facilitate separation ofcomplexes from uncomplexed forms of one or both of the proteins, as wellas to accommodate automation of the assay. Binding of a proteoglycan ofthe invention to a PT-binding partner, can be accomplished in any vesselsuitable for containing the reactants. Examples include microtitreplates, test tubes, and micro-centrifuge tubes. In one embodiment, afusion protein can be provided which adds a domain that allows theprotein to be bound to a matrix. For example,glutathione-S-transferase/ACE-2 (GST/proteoglycan of the invention)fusion proteins can be adsorbed onto glutathione sepharose beads (SigmaChemical, St. Louis, Mo.) or glutathione derivatized microtitre plates,which are then combined with the PT-inding partner, e.g. an ³⁵S-labeledPT-binding partner, and the test compound, and the mixture incubatedunder conditions conducive to complex formation, e.g. at physiologicalconditions for salt and pH, though slightly more stringent conditionsmay be desired. Following incubation, the beads are washed to remove anyunbound label, and the matrix immobilized and radiolabel determineddirectly (e.g. beads placed in scintillant), or in the supernatant afterthe complexes are subsequently dissociated. Alternatively, the complexescan be dissociated from the matrix, separated by SDS-PAGE, and the levelof proteoglycan of the invention or PT-binding partner found in the beadfraction is quantitated from the gel using standard electrophoretictechniques such as described in the appended examples.

Other techniques for immobilizing proteins on matrices are alsoavailable for use in the subject assay. For instance, either theproteoglycan of the invention or its cognate binding partner can beimmobilized utilizing conjugation of biotin and streptavidin. Forinstance, biotinylated proteoglycan molecules can be prepared frombiotin-NHS (N-hydroxy-succinimide) using techniques well known in theart (e.g., biotinylation kit, Pierce Chemicals, Rockford, Ill.), andimmobilized in the wells of streptavidin-coated 96 well plates (PierceChemical). Alternatively, antibodies reactive with the proteoglycan ofthe invention can be derivatized to the wells of the plate, and theproteoglycan of the invention trapped in the wells by antibodyconjugation. As above, preparations of a PT-binding protein and a testcompound are incubated in the proteoglycan presenting wells of theplate, and the amount of complex trapped in the well can be quantitated.Exemplary methods for detecting such complexes, in addition to thosedescribed above for the GST-immobilized complexes, includeimmunodetection of complexes using antibodies reactive with thePT-binding partner, or which are reactive with protein and compete withthe binding partner; as well as enzyme-linked assays which rely ondetecting an enzymatic activity associated with the binding partner,either intrinsic or extrinsic activity. In the instance of the latter,the enzyme can be chemically conjugated or provided as a fusion proteinwith the PT-binding partner. To illustrate, the PT-binding partner canbe chemically cross-linked or genetically fused with horseradishperoxidase, and the amount of polypeptide trapped in the complex can beassessed with a chromogenic substrate of the enzyme, e.g.3,3′-diamino-benzadine terahydrochloride or 4-chloro-1-napthol.Likewise, a fusion protein comprising the polypeptide andglutathione-S-transferase can be provided, and complex formationquantitated by detecting the GST activity using1-chloro-2,4-dinitrobenzene (Habig et al (1974) J Biol Chem 249:7130).

For processes which rely on immunodetection for quantitating one of theproteins trapped in the complex, antibodies against the proteoglycan ofthe invention, can be used. Alternatively, the protein to be detected inthe complex can be “epitope tagged” in the form of a fusion proteinwhich includes, in addition to the sequence of the core of theproteoglycan of the invention, a second polypeptide for which antibodiesare readily available (e.g. from commercial sources). For instance, theGST fusion proteins described above can also be used for quantificationof binding using antibodies against the GST moiety. Other useful epitopetags include myc-epitopes (e.g., see Ellison et al. (1991) J Biol Chem266:21150-21157) which includes a 10-residue sequence from c-myc, aswell as the pFLAG system (International Biotechnologies, Inc.) or thepEZZ-protein A system (Pharmacia, N.J.).

Cell-free assays can also be used to identify compounds which interactwith a proteoglycan of the invention and modulate an activity of aproteoglycan of the invention. Accordingly, in one embodiment, aproteoglycan of the invention is contacted with a test compound and thecatalytic activity of the proteoglycan of the invention is monitored. Inone embodiment, the ability of the proteoglycan of the invention to bindto a binding partner is determined. The binding affinity of aproteoglycan of the invention to a binding partner can be determinedaccording to methods known in the art.

B. Cell Based Assays

Cell based assays can be used, in particular, to identify compoundswhich modulate expression of a gene encoding a proteoglycan of theinvention, modulate translation of the mRNA encoding a proteoglycan ofthe invention, modulate the posttranslational modification of the coreprotein of the proteoglycan, or which modulate the stability of the mRNAor protein. Accordingly, in one embodiment, a cell which is capable ofproducing a proteoglycan of the invention, e.g., a muscle cell, isincubated with a test compound and the amount of proteoglycan of theinvention produced in the cell medium is measured and compared to thatproduced from a cell which has not been contacted with the testcompound. The specificity of the compound vis a vis the proteoglycan ofthe invention can be confirmed by various control analysis, e.g.,measuring the expression of one or more control genes.

Cell based assays can also rely on a reporter gene system detectingwhether two molecules interact or not, e.g., the classic two hybridsystem, that can be conducted in yeast or in mammalian cells.

Compounds which can be tested include small molecules, proteins, andnucleic acids. In particular, this assay can be used to determine theefficacity of antisense molecules or ribozymes that bind to RNA encodingthe proteoglycan of the invention.

In another embodiment, the effect of a test compound on transcription ofa gene encoding a proteoglycan is determined by transfection experimentsusing a reporter gene operatively linked to at least a portion of thepromoter of a gene encoding a proteoglycan of the invention. A promoterregion of a gene can be isolated, e.g., from a genomic library accordingto methods known in the art. Promoters of genes encoding proteoglycans,e.g., biglycan, are publicly available, e.g, from GenBank. The reportergene can be any gene encoding a protein which is readily quantifiable,e.g, the luciferase or CAT gene, well known in the art.

This invention further pertains to novel agents identified by theabove-described screening assays and uses thereof for treatments asdescribed herein.

C. Assays for Identifying Compounds which Modulate Phosphorylation

Biglycan was shown to bind and activate MuSK and induce phosphorylationof α-sarcoglycan. Accordingly, compounds which stimulate phosphorylationof such substrates may exercise at least part of the activity ofbiglycan in stabilizing muscle cell membranes or of potentiatingpostsynaptic membranes. Thus, also within the scope of the invention aremethods for identifying such compounds. In one embodiment, the methodcomprises contacting a cell, e.g., a muscle cell, with a compound, andmonitoring the level of phosphorylation of a DAPC component, such asα-sarcoglycan, or activation of MuSK, wherein a higher level ofphosphorylation relative to that in an untreated cell indicates that thecompound stimulates phosphorylation. Such assays can also be conductedin vitro using cell extracts or purified proteins. For example, themethod may comprise contacting a purified sarcoglycan or MuSK and a cellextract from biglycan-activated cells (i.e., cells contacted withbiglycan) or a kinase in the presence of a test compound, and monitoringwhether the presence of the test compound prevents or stimulatesphosphorylation.

VII. Additional Exemplary uses for the Proteoglycans of the Invention

The proteoglycans, or the core thereof, of the invention, can also beused as a supplement to a cell or tissue culture (e.g., system forgrowing organs). Any cell type may benefit from these supplements. Theamount of compound to be added to the cultures can be determined insmall scale experiments, by, e.g., incubating the cells or organs withincreasing amounts of a specific compound of the invention. Preferredcells include eukaryotic cells, e.g., muscle cells or neuronal cells.

Other preferred tissues include atrophic tissue. Thus, such tissue canbe incubated in vitro with an effective amount of a compound of theinvention to reverse tissue atrophy. In one embodiment, atrophic tissueis obtained from as subject, the tissue is cultured ex vivo with acompound of the invention in an amount and for a time sufficient toreverse the tissue atrophy, and the tissue can then be readminstered tothe same or a different subject.

Alternatively, the compounds of the invention can be added to in vitrocultures of cells or tissue obtained from a subject having a musculardystrophy, or other disease that can be treated with a compound of theinvention, to improve their growth or survival in vitro. The ability tomaintain cells, such as brain cells or muscle cells from subjects havinga muscular dystrophy or other disease, is useful, for, e.g., developingtherapeutics for treating the disease.

VIII. Kits of the Invention

The invention provides kits for diagnostic tests or therapeuticpurposes.

The materials for performing the diagnostic assays of the presentinvention can be made available in a kit and sold, for example, tohospitals, clinics and doctors. A kit for detecting altered expressionand/or localization of the proteoglycan of the invention, for example,can contain a reagent such as antibody binding to the proteoglycan ofthe invention, and, if desired, a labeled second antibody, a suitablesolution such as a buffer for performing, for example, animmunohistochemical reaction and a known control sample for comparisonto the test sample.

A kit for detecting altered expression of mRNA encoding the proteoglycanof the invention in a sample obtained from an individual, e.g., anindividual suspected of being predisposed to a disorder, e.g., amuscular, neuromuscular, or neurological disorder, also can be prepared.Such a kit can contain, for example one or more of the followingreagents: a reagent such as an oligonucleotide probe that hybridizes tomRNA encoding the proteoglycan of the invention, suitable solutions forextracting mRNA from a tissue sample or for performing the hybridizationreaction and a control mRNA sample for comparison to the test sample,and a series of control mRNA samples useful, for example, forconstructing a standard curve.

Such diagnostic assay kits are particularly useful because the kits cancontain a predetermined amount of a reagent that can be contacted with atest sample under standardized conditions to obtain an optimal level ofspecific binding of the reagent to the sample. The availability ofstandardized methods for identifying an individual predisposed to adisorder, e.g., muscular dystrophy will allow for greater accuracy andprecision of the diagnostic methods.

Kits for therapeutic or preventive purposes can include a therapeuticand optionally a method for administering the therapeutic or buffernecessary for solubilizing the therapeutic.

The present invention is further illustrated by the following exampleswhich should not be construed as limiting in any way. The contents ofall cited references (including literature references, issued patents,published patent applications as cited throughout this application arehereby expressly incorporated by reference.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of cell biology, cell culture,molecular biology, transgenic biology, microbiology, recombinant DNA,and immunology, which are within the skill of the art. Such techniquesare explained fully in the literature. See, for example, MolecularCloning A Laboratory Manual, 2^(nd) Ed., ed. by Sambrook, Fritsch andManiatis (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning,Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide Synthesis (M.J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683,195; Nucleic AcidHybridization (B. D. Hames & S. J. Higgins eds. 1984); Transcription AndTranslation (B. D. Hames & S. J. Higgins eds. 1984); Culture Of AnimalCells (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells AndEnzymes (IRL Press, 1986); B. Perbal, A Practical Guide To MolecularCloning (1984); the treatise, Methods In Enzymology (Academic Press,Inc., N.Y.); Gene Transfer Vectors For Mammalian Cells (J. H. Miller andM. P. Calos eds., 1987, Cold Spring Harbor Laboratory); Methods InEnzymology, Vols. 154 and 155 (Wu et al. eds.), Immunochemical MethodsIn Cell And Molecular Biology (Mayer and Walker, eds., Academic Press,London, 1987); Handbook Of Experimental Immunology, Volumes I-IV (D. M.Weir and C. C. Blackwell, eds., 1986); Manipulating the Mouse Embryo,(Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1986).

IX. EXAMPLES Example 1 Characterization of a Dystroglycan-BindingProtein, DAG-125

This Example describes the identification of a dystroglycan-bindingprotein, termed DAG-125.

In order to identify novel dystroglycan binding partners, a ligand blotoverlay assay, was developed as follows. Postsynaptic and non-synapticmembrane fractions from Torpedo electric organ were prepared aspreviously described (Bowe, et al. (1994) Neuron. 12: 1173). Allhandling of membranes and protein was performed at 4° C.

Membrane proteins were separated by SDS-PAGE (5-15% gradient gel), andtransferred to nitrocellulose. To detect dystroglycan binding proteins,the nitrocellulose was rinsed and blocked for 3 hr in Hank's BalancedSalt Solution containing 1 mM CaCl₂, 1 mM MgCl₂, 1% bovine serumalbumin, 1% Nonfat Dry Milk, 1 mM DTT, 10 mM HEPES, pH 7.4, and was thenincubated overnight in the same buffer containing³⁵S-methionine-labelled dystroglycan fragments produced by in vitrotranscription/translation as follows.

DNA fragments encoding DG₁₋₈₉₁ and DG₃₄₅₋₈₉₁ (human alpha-dystroglycansequence is described, e.g., in Ibraghimov-BeskrovnayaHum (1993) MolGenet 2: 1651) were cloned in the in vitro expression vector pMGTdeveloped by A. Ahn (Ahn and Kunkel (1995) J Cell Biol. 128: 363).Additional in vitro expression plasmids used in this study (includingDG₁₋₇₅₀, DG₇₇₆₋₈₉₁, and DG₃₄₅₋₆₅₃) were prepared by PCR-based subcloningof these inserts. The PCR primers included restriction sites forreligation into the EcoRI site of pMGT. Dystroglycan protein fragmentswere generated by in vitro transcription/translation using the PromegaTNT T7 coupled reticulocyte system as per the manufacturer'sinstructions. For protein to be used in ligand blot overlay assay, thereaction mixture contained ³⁵S-methionine (with no unlabeledmethionine). After incubation for 2 hr, the reaction mixture was passedover Bio-Spin desalting columns (Bio-Rad, Hercules, Calif.) to removeunincorporated amino acids and salts.

After incubation of the blots with the in vitro translated proteins, theblots were rinsed and dried and bound dystroglycan fragments werevisualized by autoradiography. To detect dystroglycan present in theSDS-PAGE sample, an agrin blot overlay assay was performed essentiallyas described in O'Toole, et al. (1996) PNAS 93:7369. Briefly, thenitrocellulose was rinsed and blocked for 3 hr in HEPES-buffered MinimumEssential Medium supplemented with 1% bovine serum albumin and 10% horseserum. It was then incubated for 4 hr in this buffer containingrecombinant rat agrin (isoform A₀B₀, prepared as described in O'Toole etal., supra), followed by a second layer containing 1 μg/ml anti-agrinantibody ¹²⁵I-Mab-131 (Stressgen Laboratories, Victoria, BC). Boundanti-agrin antibody was visualized by autoradiography.

The results are shown in FIG. 2. Lanes 1 and 2 indicate that certainfragments of dystroglycan bound to an about 125 kD, highly glycosylatedpolypeptide, which was termed DAG-125 (for “Dystroglycan-AssociatedGlycoprotein, 125 kDa”). As shown in FIG. 2A, the extracellular domainof dystroglycan (lane 1: DG₁₋₇₅₀) bound to DAG-125, while theintracellular portion of dystroglycan (lane 2: DG₇₇₆₋₈₉₁) did not.

Lanes 3 and 4 of FIG. 2 show that DAG-125 is enriched in synaptic ascompared to non-synaptic membranes.

To solubilize DAG-125, synaptic membranes were centrifuged at 100,000×gfor 1 hour (hr) and resuspended in ddH₂O. The pH was adjusted to 11.0 or12.0 (as indicated) with NaOH and the membranes stirred for 1 hr.Insoluble material was removed by centrifugation at 100,000×g for 1 hr.The alkaline extract was neutralized with 10 mM Tris HCl and adjusted topH 7.4. DAG-125 remained soluble under these conditions as determined byresistance to pelleting during a second centrifugation. Lanes 5-7 ofFIG. 2 show that DAG-125 is a peripheral membrane protein that can beextracted from the synaptic membrane by alkaline treatment. Synapticmembranes were extracted at pH 12 and the insoluble (lane 6) and solublefraction (lane 7) were analyzed. Greater than 90% of DAG-125 issolubilized by pH 12.0 treatment. Thus, DAG-125 is likely to be aperipheral membrane protein, since it is removed from the membranes byalkaline-treatment.

Example 2 Association Between α-Dystroglycan and DAG-125

This Example demonstrates that DAG-125 associates with invitro-translated α-dystroglycan, bacterially produced GST-α-dystroglycanfusion protein and native α-dystroglycan in solution.

DAG-125 was solubilized by alkaline-treatment, and neutralized, asdescribed above, and incubated with column matrices and recombinant ornative dystroglycan as indicated in FIG. 3. The input material andeluates from the beads were analyzed by ligand blot overlay assay forthe presence of DAG-125 (³⁵S-DG345-653 as probe) or nativeα-dystroglycan (agrin overlay, see Example 1).

FIG. 3A shows DAG-125 incubated with goat anti-mouse Ig-conjugatedagarose beads in the presence or absence of in vitro translateddystroglycan polypeptide (DG₃₄₅₋₇₅₀) and/or anti-dystroglycan monoclonalantibody (NCL-β-DG; Novocastra, Newcastle-on-Tyne, UK). The resultsindicate that DAG-125 co-precipitated with dystroglycan plusanti-dystroglycan antibody (lane 5), but was not precipitated in theabsence of either or both (lanes 2-4). Thus, DAG-125 binds to in vitrotranslated dystroglycan peptide DG345-750.

FIG. 3B shows DAG-125 incubated with glutathione-sepharose beads thathad been pre-incubated with either bacterially produced GST or abacterially produced GST-dystroglycan fusion protein (GST-DG₃₄₅₋₆₅₃). Afusion protein of glutathione S-transferase (GST) and amino acids345-653 of dystroglycan was produced by using PCR-based subcloning tointroduce dystroglycan coding sequence into the bacterial proteinexpression vector pGEX-1 T (Pharmacia, Piscataway, N.J.). The resultingbacterial expression plasmid, pGST-DG₃₄₅₋₆₅₃, was then introduced intothe E. coli strain BL21 and expressed fusion protein recovered from thecytoplasmic fraction as per manufacturer's instructions. Control protein(GST) was obtained using pGEX-1 T. The results show that DAG-125 wasco-precipitated with the dystroglycan fusion protein (lane 3), but notwith GST alone (lane 2). Thus, DAG-125 binds to alpha-dystroglycanpeptide 345-653 produced in bacteria.

FIG. 3C shows DAG-125 and native α-dystroglycan. Alkaline extracts ofTorpedo electric organ membranes contain both DAG-125 andα-dystroglycan. This extract was applied to agarose columns conjugatedto either control antibody or to an anti-Torpedo dystroglycan monoclonalantibody (MAb3B3; Bowe, M. A., et al. (1994) Neuron. 12: 1173). Theresults show that native α-dystroglycan and DAG-125 were co-precipitatedby the anti-Torpedo dystroglycan antibody, Mab3B3, (lanes 3 and 6), butnot by control antibody (lanes 2 and 5). Western blots indicate thatMab3B3 does not recognize DAG-125 (see Bowe, M. A., et al., 1994,Neuron. 12: 1173-1180).

Thus, FIG. 3 shows that DAG-125 co-precipitates with in vitro-translatedalpha-dystroglycan, bacterially produced GST-alpha-dystroglycan protein,and with native alpha-dystroglycan.

Example 3 Localization of the DAG-125 Binding Domain of α-Dystroglycan

This Example describes that the DAG-125 binding domain of α-dystroglycanis contained in an approximately 150 amino acid carboxyl-terminal domainof the protein.

In order to determine the region of α-dystroglycan that interacts withDAG-125, a panel of dystroglycan fragments were prepared by in vitrotranslation (FIG. 4) and the ability of each to bind DAG-125 was testedusing the ligand blot overlay assay. FIG. 4, which show the results,indicates that DAG-125 binds to the carboxyl-terminal one-third ofα-dystroglycan. A small contribution from the middle third ofα-dystroglycan is also possible. The ectodomain of β-dystroglycan doesnot appear to contribute to binding of DAG-125. Moreover, thesefragments were produced under conditions in which the polypeptides arenot glycosylated. Therefore, carbohydrate side chains on dystroglycanare not necessary for its binding to DAG-125.

Thus, the major binding domain is contained in about 150 amino acidregion of dystroglycan. The location of this domain and the lack of acarbohydrate requirement indicate that α-dystroglycan's binding site forbiglycan is distinct from that mediating association with agrin,laminin, and perlecan.

Example 4 Identification of DAG-125 as Biglycan or a ProteoglycanRelated Thereto

This Examples demonstrates that DAG-125 is biglycan or a protein relatedthereto.

It was found that DAG-125 co-purified with postsynaptic membranes, butthat, however, it was insoluble in all non-ionic detergents testedincluding Triton X-100 and n-octyl-β-D-glucopyranoside, both of whichefficiently extract α/β-dystroglycan from these membranes (Bowe, et al.(1994) Neuron. 12: 1173; Deyst, et al. (1995) J Biol Chem. 270:25956-9). Even without detergent, about 50% of DAG-125 could beextracted at pH 11 and near-complete solubilization was achieved by ashort pH 12 treatment (see FIG. 2A). Importantly, DAG-125 remainedsoluble when returned to neutral pH. Based upon these properties and thefindings that DAG-125 binds to both heparin and chondroitin sulfatecolumns, the following purification protocol was developed.

Postsynaptic-rich membrane fractions were first pre-extracted with 25 mMn-octyl-D-glucopyranoside to remove detergent-soluble proteins. DAG-125was then solubilized by alkaline extraction (pH 12.0), as described inExample 1. The alkaline extract was diluted in SEN Buffer (20 mM TrisHCl, 100 mM NaCl, 23 μg/ml aprotinin, 0.5 μg/ml leupeptin, 5 mMbenzamidine, 0.7 μg/ml pepstatin A, 1 mM phenylmethylsulfonylflouride,0.02% azide, 0.1% Tween 20, pH 7.6) and recentrifuged to remove anyproteins precipitating upon neutralization. The extract remained in SENBuffer for the remainder of the purification, with only the NaClconcentration changed as indicated. The extract was passed over a MAb3B3column (Bowe, et al. (1994) Neuron. 12: 1173) to remove α-dystroglycan.The MAb3B3 column flow-through was passed over a combined,non-DAG-125-binding lectin-agarose column (peanut agglutinin and ulexeuropaeus agglutinin I, Vector Labs, Burlingame, Calif.) as a secondpre-clear. The flow-through was next applied to a column of chondroitinsulfate-agarose (CS-agarose). The CS-agarose column was prepared bycoupling chondroitin sulfate B (Sigma, St. Louis, Mo.; #C-3788) to-aminohexyl-agarose (Sigma) activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide (Sigma). Afterincubation with the lectin column flow-through, the CS column was washedextensively and eluted with a 0.1-2.0 M NaCl gradient. DAG-125 eluted in0.3-0.65 M NaCl. These fractions were pooled, diluted to 0.3 M NaCl, andapplied to a heparin-agarose column (Sigma #H-0402). The column waswashed and eluted with a 0.3-2 M NaCl gradient. DAG-125 eluted in0.6-0.85 M NaCl. These fractions were pooled, concentrated by ethanolprecipitation (final purity of DAG-125 of about 30%), redissolved inSDS-PAGE sample buffer, separated on a 5-15% gradient gel, andtransferred to a PVDF membrane. A portion of the PVDF membrane wasanalyzed for DAG-125 by blot overlay and the remainder was transientlystained with Ponceau S. Two regions (“U” and “L”; see FIG. 5A) of theDAG-125 band on the Ponceau stained membrane were excised and digestedwith trypsin. The released peptides were analyzed by HPLC using a C8column and UV detection. The column profiles were virtually identical,indicating that the polydisperse band is due to the presence of asingle, heterogeneously glycosylated protein.

Three peptides from the trypsin digest were collected as fractions fromthe HPLC analysis and subjected to automated Edman degradation, asdescribed previously (Bowe, et al. (1994) Neuron. 12: 1173). Thesequences obtained were compared to public databases. The alignment ofthe Torpedo DAG-125 peptides to the deduced sequence of human biglycan(amino acids 241-249; 258-266; and 330-348) is shown in FIG. 5B. Humanbiglycan is described in Fisher et al. (1989), infra) and its amino acidsequence is set fort in SEQ ID NO: 9. All DAG-125 peptide fragments werehighly homologous to mammalian biglycan, with an overall 76% identity(FIG. 5B). Thus, DAG-125 is a Torpedo orthologue of mammalian biglycanor a close homolog thereof.

Human biglycan, produced in the vaccinia system, as described below, wasalso shown to bind to α-dystroglycan. The binding was less strong thanwith Torpedo DAG-125, probably reflecting the fact that the biglycanproduced in this system is a mixture of core biglycan and proteoglycanbiglycan. However, this further supports that Torpedo orthologue ofmammalian biglycan or a close homolog thereof.

The domain structure of human biglycan is shown in FIG. 5C. Biglycan isone of a family of small leucine-rich repeat proteins (Hocking et al.(1998) Matrix Biol. 17: 1). It consists of a pre-pro-peptide that is notpresent in the mature polypeptide. This domain is followed by a shortunique sequence with two chondroitin sulfate attachment sites (shown asstacked beads in the Figure). There are two pairs and one pair ofdisulfide-linked cysteines at the amino and carboxyl-terminal domains,respectively. Finally, the bulk of the protein is comprised of 10 (or 11depending upon the classification of the region within thecarboxyl-terminal cysteine pair) leucine-rich repeats. The position ofthe three Torpedo peptides relative to the human sequence is indicatedby horizontal lines.

Example 5 Chondroitin Sulfate Chains of Biglycan are Necessary forBinding of Biglycan to α-dystroglycan

Mammalian biglycan is often substituted with chondroitin sulfate. Todetermine if Torpedo biglycan is also a chondroitin sulfate proteoglycanand whether glycosylation is important for its binding toα-dystroglycan, DAG-125 was digested with various glycosidases andglycosaminoglycanases and the products were analyzed by α-dystroglycanligand blot overlay with ³⁵S-DG345-653.

Enzyme treatments were carried out on alkaline-extracted Torpedoelectric organ synaptic membrane proteins at 37° C. overnight. Enzymes,final concentration, supplier and catalog numbers are listed in Table I.All reactions were performed in the protease inhibitors present in SENBuffer, with the addition of 1 mM EDTA, 10 mM N-ethylmaleimide, and 0.8%mouse serum albumin. Chondroitinases (all forms) were buffered with 100mM Tris-acetate (pH 8.0). Hyaluronidase and keratanase were bufferedwith 50 mM sodium acetate (pH 5.0). Heparinases (I, II, and III),chondro-4-sulfatase and chondro-6-sulfatase were buffered with 10 mMNaPO₄ (pH 7.4). N-Glycanase, O-glycanase, neuraminidase,α-N-acetylgalactosaminidase, β-N-acetylglucoasaminidase were bufferedwith 50 mM Tris HCl (pH 7.3). Control treatments included buffers andprotease inhibitors without added enzymes.

The results, are shown in FIG. 6 and in Table I.

TABLE I Enzyme Inhibit (Units/ Enzyme Binding? mL) ConcSource Cat. #Chondroitinase ABC + 0.5 Sigma C-2905 Chondroitinase ABC + − 0.5 SigmaC-2905 5 mM ZnCl₂ Chondroitinase ABC, + 0.5 Sigma C-3667 Protease-freeChondroitinase ABC, + 0.5 Roche 1080717 Protease-free ChondroitinaseAC + 0.5 Sigma C-2780 Chondroitinase B +/− 25 Sigma C-8058 Heparinase I− 25 Sigma H-2519 Heparinase II − 5 Sigma H-3812 Heparinase III − 5Sigma H-8891 (Heparitinase) Chondro-4-sulfatase +/− 0.5 Sigma C-2655Chondro-6-sulfatase − 0.5 Sigma C-2655 Keratanase − 0.02 Roche 982954α-N- − 2 Sigma A-9763 acetylgalactosaminidase β-N- − 8 Sigma A-2264acetylglucoasaminidase N-Glycanase − 15 Genzyme N-Gly-1 O-Glycanase −0.03 Genzyme B2950 Neuraminidase − 1 Genzyme NSS-1

The results indicate that removal of chondroitin sulfate side chainsabolished the binding to α-dystroglycan. Chondroitinase B (specific fordermatan sulfate) had a much smaller effect compared to chondroitinaseswhich removed chondroitin sulfate A and C. No other glycosidase orglycosaminoglycanase treatment had a detectable effect on α-dystroglycanbinding (see Table I). Several lines of evidence indicate that theeffects of chondroitinase digestion are due to chondroitinase activityand not to contaminating proteases: 1) the digestions were performed ina cocktail of protease inhibitors; 2) the same result was seen with fourdifferent preparations of chondroitinase, including two which had beenaffinity purified to remove proteases; and 3) the effect was preventedby addition of 5 mM Zn²⁺, an inhibitor of chondroitinase but not ofproteases.

To further investigate the binding properties of biglycan, the bindingof α-dystroglycan to biglycan derived from a variety of sources, as wellas to decorin, a small leucine-rich proteoglycan that is about 50%identical to biglycan, were investigated.

Biglycan (or decorin) were analyzed by SDS-PAGE and Coomassie BrilliantBlue staining for protein (lanes 1-5 of FIG. 7) or blot overlay assayfor dystroglycan binding (lanes 6-10 of FIG. 7): lanes 1, 6: alkalineextract of Torpedo synaptic membranes (1 μg total protein, of whichbiglycan is estimated to be <2%); lanes 2, 7: lysate of non-inducedbacteria; lanes 3, 8: lysate of induced bacteria expressing recombinanthuman biglycan (QE-Bgn; prominent band at ˜37kD—arrow); lanes 4, 9:biglycan purified from bovine articular cartilage (4 μg; Sigma); lanes5, 10: decorin purified from bovine articular cartilage (4 μg; Sigma).The results indicate that biglycan present in electric organ bindsdystroglycan much more strongly then biglycan or decorin purified fromarticular cartilage (compare Coomassie staining to dystroglycanoverlay).

The recombinant human biglycan was produced as follows. P16, a cloningplasmid consisting of Bluescript containing a cDNA encoding humanbiglycan (SEQ ID NO: 9) was provide by Larry Fisher (National Instituteof Dental Research, National Institutes of Health) (Fisher et al.(1989), supra). The sequence encoding the mature secreted peptide (aminoacids 1-343) was amplified by PCR and subcloned into the bacterialexpression vector pQE9 (Qiagen, Valencia, Calif.). The resultingplasmid, pQE-biglycan, adds the sequence MRGSHHHHHHGS (SEQ ID NO: 10) tothe amino terminus Recombinant protein was produced in E. coli strainM15[pREP4]. Uninduced bacteria provide control protein. Induced ornon-induced bacteria were isolated by cnetrifugation and resuspended inSDS-PAGE sample buffer for analysis by ligand blot overlay. Thus,bacterially-expressed biglycan, which contains no chondroitin sulfateside chains, did not bind α-dystroglycan (FIG. 7), consistent with arequirement for chondroitin sulfate chains. Biglycan purified fromarticular cartilage bound α-dystroglycan poorly, even at >100-foldhigher loading than that used for Torpedo biglycan analysis. Thesefindings indicate that specific chondroitin sulfate chains are requiredto mediate α-dystroglycan binding to biglycan.

Thus, biglycan from Torpedo synaptic membranes is substituted withchondroitin sulfate chains, which are predominantly chondroitin sulfateA and/or C, and chondroitin sulfate substitution of biglycan isnecessary for binding to dystroglycan.

Example 6 Biglycan Binds to Sarcoglycan Components

This Example describes that biglycan core binds to α- and to gammasarcoglycans and that biglycan proteoglycan also binds to γ-sarcoglycan,and that decorin failed to bind to any of the sarcoglycans (nodetectable level of binding was observed).

The binding of biglycan and decorin to the different components ofsarcoglycan of the DAPC was investigated by overlay assay usingrecombinantly produced human sarcoglycans, on biglycan proteoglycan(core and side chains), biglycan core (no side chains), decorinproteoglycan (core and side chains), decorin core (no side chains), ahybrid between biglycan and decorin core (the “hybrid” with sidechains), and Torpedo electric organ membrane fraction (TEOM). The hybridcontained the first 30 amino acids of human biglycan (cysteine richdomain) and the remaining portion of the biglycan molecule was swappedwith that of decorin. The sarcoglycans were produced by in vitrotranscription and translation using a Promega TNT kit, as described inAhn and Kunkel (1995) J. Cell Biol. 128: 363. The biglycan and decorincore polypeptide and proteoglycan were produced recombinantly byvaccinia-virus infection of rat osteosarcoma cells, as described inHocking et al. (1996) J. Biol. Chem. 271:19571. Briefly, the cDNAsequence encoding the mature core protein of human biglycan ligated to apolyhistidine fusion cassette under the control of T7 promoter wasinserted into the pBGN4 vector. An encephalomyocarditis virusuntranslated region was inserted downstream of the T7 promoter tofacilitate cap-independent ribosome binding and thereby increasestranslation efficiency up to 10-fold. The fusion cassette encodes thecanine insulin signal sequence (INS), six consecutive histidine residues(POLYHIS), and the factor Xa recognition site (Xa). A recombinantvaccina virus, vBGNA, encoding the T7 regulated BGN4 construct, wasgenerated by a homologus recombination event between wild-type vacciniavirus and thymidine kinase flanking sequences in the plasmid, pBGN4.There are two extra amino acids between the polyhistidine sequence andthe Factor Xa site and two extra amino acids between the Factor Xa siteand the start of the mature core protein sequence of biglycan. Thus, thevector contains from 5′ to 3′: EMCUTR-INS-POLYHIS-[Glu-Ser]-Xa-[Leu-Glu]-mature biglycan devoid of thebiglycan signal sequence and propeptide sequence). The biglycan that isproduced from this system is a mixture containing proteoglycan biglycanand biglycan devoid of glycaosaminoglycan chains (“core biglycan”).

The overlay assays were preformed as described above for DAG-125.

The results, which are shown as FIGS. 8A-C, indicate the following:α-sarcoglycan binds to biglycan core and to the hybrid; γ-sarcoglycanbinds to biglycan core, to biglycan proteoglycan and very weakly to thehybrid; and δ-sarcoglycan binds to biglycan core very weakly.

Thus, biglycan binds to -sarcoglycan via its core peptide. Furthermore,since the hybrid binds to -sarcoglycan, but that decorin does not bindto it, binding of biglycan to α-sarcoglycan occurs through theN-terminal 30 amino acids of biglycan, i.e., the region that includesthe cysteine-rich region, but no leucine-rich repeats. In addition, theresults indicate that glycosylation of sarcoglycan is not necessary forits binding to biglycan.

Human biglycan was also shown to bind to native α- and γ-sarcoglycan insolution. This was demonstrated by isolating native human α- andγ-sarcoglycan by detergent extraction of cultured mouse myotubes,incubating the extracts with recombinant human core biglycan prepared asdescribed above, and then immumoprecipitating the resulting complexeswere then immunoprecipitated with antibodies to α-sarcoglycan (vectorlaboratories). The immunoprecipitates were then resolved bysds-polyacrylamide gel electrophoresis and western blotted withantibodies to biglycan. Tthe anti-biglycan antibody was raised against abacterially-produced biglycan fusion protein. The results, which areshown in FIG. 8D, show that native sarcoglycans alpha and gamma bind tobiglycan.

Example 7 Biglycan is Expressed at Synaptic and Non-Synaptic Regions andis Up-Regulated in Dystrophic Muscle

Previous reports have shown that biglycan mRNA and protein are expressedin muscle (Bianco, et al. (1990) J. Histochem Cytochem. 38: 1549; Bosse,et al. (1993) J. Histochem. Cytochem. 41: 13). Since the biglycan thatwas used in the above-described Examples was obtained from synapticmembranes, it was investigated whether biglycan is also expressed at theneuromuscular junction.

Frozen sections of normal adult mouse muscle were double-labeled withα-bungarotoxin (Bgtx; to localize AChRs) and antibodies to biglycan.Cryostat sections (10 μm) of leg muscle from fresh-frozen wild-type (C57BL) mice were mounted on slides, fixed, and treated with chondroitinaseessentially as described in (Bianco, P., et al., 1990, J HistochemCytochem. 38:1549). Primary antibodies were anti-biglycan (LF-106;generously provided by L. Fisher) diluted in PBS containing 5% BSA, 1%normal goat or horse serum, and 0.1% Triton X-100. Incubation in primaryantibodies or non-immune control serum proceeded overnight at 4° C.Except where noted, all subsequent steps were performed at roomtemperature. Bound antibodies were detected with Cy3-labelledanti-rabbit Ig (Jackson Laboratories, West Grove, Pa.). Fordouble-labelling, sections were first fixed for 5 min in 1%formaldehyde, rinsed and incubated in fluorescein-conjugatedα-bungarotoxin (Molecular Probes, Eugene Oreg.) for 1 hr. The sectionswere then washed, fixed, treated with chondroitinase and stained forbiglycan as described above. Sections were air-dried, mounted inCitifluor (Ted Pella, Redding, Calif.) and examined on a Nikon Eclipsemicroscope. Images were acquired on a cooled CCD camera using IP LabSpectrum software and then imported to Adobe Photoshop.

The results, which are shown in FIG. 9, indicate that biglycanimmunoreactivity is distributed over the entire periphery of themyofibers and synapses, and that it is also concentrated at someneuromuscular junctions.

Since biglycan binds to a component of the DAPC, it was investigatedwhether or not its expression was altered in a mouse model of musculardystrophy in which dystrophin is absent, i.e., the mdx mouse. Adultmice, which contain almost exclusively regenerated muscle fibers thatsurvive due to utrophin compensation were investigated (Grady, et al.(1997) Cell 90: 729). Frozen sections of normal and mdx muscle from 6 wkold mice were mounted on the same slides and immunostained for biglycanas described above. Immunostaining revealed that the level of biglycanexpressed in mdx muscle is elevated compared to control animals (FIG.10). These observations raise the possibility that biglycan could bepart of the compensatory mechanism that allows survival of dystrophinnegative muscle fibers.

Example 8 Biglycan Binds to the MuSK Ectodomain

This Example demonstrates that biglycan binds to other components of thesynaptic membrane, in particular, the MuSK ectodomain.

Torpedo biglycan (DAG-125) was solubilized by alkaline extraction andneutralized, as described in Example 1, and incubated with proteinA-agarose beads and with either human IgG (HIgG) or with human Fc fusionproteins containing the ectodomains of recombinant human MuSK (Glass etal. (1996) Cell; and Donzuela et al. (1995) Neuron), TIE-2, or TRK forco-precipitations. The results, which are shown in FIG. 11, indicatethat Torpedo biglycan binds to the MuSK ectodomain, but not to IgG, norto the two unrelated receptor tyrosine kinase ectodomains TIE-2 and TRK.It was also shown that MuSK solubilized from muscle membranes binds toTorpedo biglycan. Decorin was also shown to bind to MuSK.

Thus, DAG-125 binds to MuSK.

Example 9 Biglycan Preparations Potentiate Agrin-Induced AChR Clusteringon Myotubes

This Example demonstrates that biglycan potentiates agrin-induced AChRclustering.

Primary chick myotubes were incubated for 20 hours with recombinantbiglycan core (no GAG) with or without the addition of 1 unit (about 10μM) of recombinant rat agrin isoform 12-4-8. Cultures incubated in 1 nMbiglycan+agrin increased AChR clustering by an average of 50% overcultures incubated in 1 unit of agrin only. Higher concentrations ofbiglycan had no effect or possibly inhibited agrin-induced clustering.In another example, exogenous biglycan-enriched preparations (about 30%pure) were also found to potentiate agrin-induced AChR clustering whenapplied to cultured chick myotubes.

Thus, biglycan potentiaties (50% increase) agrin-induced AChR clusteringwhen present at about 10⁻⁹ M (i.e., about 1.4 nM). At higherconcentrations (10⁻⁸ M, 10⁻⁷ M, i.e., about 140 nM) biglycan inhibitsagrin-induced AChR clustering. This was demonstrated on wild-type chickmyotubes, which were prepared as described in Nastuk et al., 1991(Neuron 7: 807-818), using either core or proteoglycan human recombinantbiglycan, produced by the vaccinia system, described above. Thus, thereis a biphasic effect of biglycan on agrin-induced AChR clustering.

Example 10 Biglycan and Decorin Induce Tyrosine Phosphorylation of MuSK

The culture of chick myotubes with agrin resulted, as expected, in thestimulation of phosphorylation of MuSK. It was observed that thestimulation of chick myotubes with human biglycan proteoglycan,decorin-proteoglycan, biglycan core and decorin core (separately) alsoinduce tyrosine phosphorylation of MusK on muscle cells. Phosphorylationwas determined by immunoprecipitation and Western blot using ananti-phosphotyrosine antibody. The biglycan and decorin proteoglycan andcore were produced by the vaccinia system described above. The resultsare shown in FIG. 12.

Similarly to agrin-induced AChR clustering, agrin-induced MuSKphosphorylation was also shown to be biphasic: human biglycan core caneither potentiate (at 1.4 nM) or inhibit (at 140 nM) agrin-induced MuSKphosphorylation in cultured C2C12 myotubes.

Example 11 Myotubes Cultured from Biglycan^(−/o) Mice Show a DefectiveResponse to Agrin

The role of biglycan in mediating agrin-induced AChR clustering wasfurther proved by using biglycan knockout mice (biglycan^(−/o) malemice).

Biglycan^(−/o) mice were generated by Marian Young at the NIH. PCRgenotyping of the mice was performed on genomic DNA using primer pairsspecific for mutant and wild type biglycan alleles (Xu et al. (1998)Nat. Genet. 20:78). PCR products from a wild type (male; +/o), aheterozygote (female; +/−), and a knockout (male; −/o) are shown in FIG.13A.

A Bgn female (+/−) was mated to a Bgn male (+/o) and primary cultureswere established from each male pup in the resulting litter. Thegenotype of each pup was determined as described in the previousparagraph. Myotube cultures derived from each mouse were then treatedeither with or without recombinant agrin 4.8 for 18 hours. Agrin 4.8 isan alternatively spliced variant, having a four amino acid insert atsite Y and an eight amino acid insert at site Z (see, e.g., Iozzo R. I(1998) Ann. Rev. Biochem. 67:609, and Firns et al. (1993) Neuron11:491). Myotubes were then labeled with rhodamine-bungarotoxin tovisualize AChRs. As shown in FIG. 13B, the agrin-induced AChR clusteringon the biglycan^(−/o) myotubes is greatly reduced compared to those fromwild type littermate controls. These results thus provide strong anddirect evidence for a role of biglycan in agrin-induced AChR clustering.

FIG. 13C shows a quantitation of AChR clustering. AChR clusters andmyotubes were counted in a minimum of 10 fields for cultures treatedeither with (AGRIN) or without (Con) recombinant agrin 4.8.

Example 12 Recovery of Response to Agrin in Biglycan^(−/o) Mice by theAddition of Recombinant Biglycan

This example shows that the defective response of AChR aggregation inbiglycan^(−/o) mice in response to agrin can be rescued by the additionof exogenous recombinant humanbiglycan core.

This was demonstrated by adding 1.4 nM (0.05 micrograms/ml) ofrecombinant core human biglycan, produced in the vaccinia systemdescribed above, to the cultures of biglycan^(−/o) myotubes described inExample 11. AchR clustering was measured as determined in Example 11.

The results, which are presented in FIG. 13B, indicate that the additionof biglycan core restores the response of biglycan^(−/o) myotubes toagrin.

Thus, this experiment proves the importance of biglycan in agrin-inducedAChR clustering. In addition, since this example was performed with corebiglycan, i.e., with no proteoglycan side chains, this exampledemonstrates that the core is particularly important for theagrin-induced postsynaptic differentiation. This further demonstratesthat biglycan affects a cell simply by contacting the cell withbiglycan.

Example 13 Serum Creatine Kinase is Elevated in Biglycan Knockout Mice

Serum creating kinase (CK) levels from four mice (two male, two female)ages 16 weeks old were assayed. As shown in FIG. 15, CK levels frombiglycan knockout mice are about 10 fold greater than wild types. Serafrom three other wild type female mice had similar CK levels as thesewild type males.

Thus, although biglycan^(−/o) mice do not show gross abnormalities (Xuet al. (1998) Nat. Genet. 20:78), the expression of dystrophin andutrophin are not grossly abnormal, and the synapses also appear grosslynormal, they have an abnormally high CK level, relative to wildtypeanimals. Such elevations are a hallmark of muscle cell damage, such asthat seen in muscular dystrophy (Emery (1993) Duchenne MuscularDystrophy Oxford Monographs on Medical Genetics. Oxford: New York.Oxford Univ. Press). In addition, these mice have leaky membranes, asjudged by Evans Blue uptake, and show signs of muscle cell death andregeneration as judged by the presence of myofibers withcentrally-located nuclei in the adult. Thus, these results indicate thatthe muscle cell plasma membrane is likely to be compromised in theseanimals. These observations, together with the restoration ofagrin-induced AChR clustering in myotubes from biglycan^(−/o) mice bythe addition of biglycan, strongly suggest that the absence of biglycanor the presence of a defective biglycan results in defective muscleand/or nerve plasma membrane which can be restored by the addition ofexogenous biglycan.

Example 14 Biglycan Core Stimulates MuKD Dependent TyrosinePhosphorylation of α-Sarcoglycan and a 35 kD DAPC Component in Myobtubes

This example demonstrates that biglycan induces tyrosine phosphorylationof DAPC components and has therefore a signaling function.

Human biglycan was prepared using the vaccina system described aboveWildtype myotubes or MuSK null myotubes were incubated for 30 minutes inthe presence of 1 microgram/ml (27 nM) of a mixture of core andproteoglycan forms of human biglycan. The cultures were detergentextracted and α-sarcoglycan was immunoprecipitated, separated bySDS-PAGE, blotted, and probed with anti-phosphotyrosine antibody orMIgG. The results, which are presented in FIG. 15, show that thetyrosine phosphorylation of α-sarcoglycan is increased in the presenceof biglycan in wild type cells, but not in MuSK null myotubes. Inaddition, it was observed that an unidentified 35 kD DAPC component wasalso phosphorylated in wild type cells but not in MuSK null myotubes Inaddition, the results show that biglycan is capable of a signalingfunction, in the absence of agrin.

Equivalents

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents of the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A method for stabilizingdystrophin-associated protein complexes (DAPCs) on the surface of acell, comprising contacting the cell with an effective amount of abiglycan therapeutic, such that the DAPCs are stabilized, wherein thebiglycan therapeutic is a polypeptide comprising a biglycan amino acidsequence which is at least about 90% identical to amino acids 38-365 ofSEQ ID NO:
 9. 2. The method of claim 1, wherein the biglycan therapeuticbinds to Muscle-specific kinase (MuSK) on the cell.
 3. The method ofclaim 1, wherein the biglycan therapeutic binds to a α-sarcoglycanand/or γ-sarcoglycan on the cell.
 4. The method of claim 1, wherein thebiglycan therapeutic induces phosphorylation of sarcoglycans on a cellmembrane.
 5. The method of claim 1, wherein the biglycan therapeuticupregulates utrophin levels in the cell.
 6. The method of claim 1,wherein the biglycan amino acid sequence comprises one or more motifs of24 consecutive amino acids in the Leucine Rich Repeat (LLR) of SEQ IDNO:
 9. 7. The method of claim 1, wherein the polypeptide is derivatizedwith one or more glycosaminoglycan (GAG) side chains.
 8. The method ofclaim 1, wherein the biglycan amino acid sequence is at least about 95%identical to amino acids 38-365 of SEQ ID NO:
 9. 9. The method of claim1, wherein the biglycan amino acid sequence is encoded by a nucleic acidwhich hybridizes under stringent conditions of 6.0×sodiumchloride/sodium citrate (SSC) at about 45° C. to a complementary strandof SEQ ID NO:
 8. 10. The method of claim 1, wherein the cell is a musclecell.